Middle east respiratory syndrome coronavirus immunogens, antibodies, and their use

ABSTRACT

Methods of inducing an immune response in a subject to the Middle East respiratory syndrome coronavirus (MERS-CoV) are provided. In several embodiments, the immune response is a protective immune response that inhibits or prevents MERS-CoV infection in the subject. Recombinant MERS-CoV polypeptides and nucleic acid molecules encoding same are also provided. Additionally, neutralizing antibodies that specifically bind to MERS-CoV S protein and antigen binding fragments thereof are disclosed. The antibodies and antigen binding fragments are useful, for example, in methods of detecting MERS-CoV S protein in a sample or in a subject, as well as methods of preventing and treating a MERS-CoV infection in a subject.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional of U.S. application Ser. No. 15/553,466, filed Aug.24, 2017, which is the U.S. National Stage of International ApplicationNo. PCT/US2016/019395, filed Feb. 24, 2016, which was published inEnglish under PCT Article 21(2), which in turn claims the benefit ofU.S. Provisional Application No. 62/120,353, filed Feb. 24, 2015. Eachof the prior applications is incorporated by reference herein in itsentirety.

FIELD OF THE DISCLOSURE

This disclosure relates to recombinant Middle East respiratory syndromecoronavirus (MERS-CoV) polypeptides, immunogenic fragments thereof, andmonoclonal antibodies specific for same, for treatment and prevention ofMERS-CoV infection and disease.

BACKGROUND

MERS-CoV has emerged as a highly fatal cause of severe acute respiratoryinfection. Thousands of infections and hundreds of deaths have beenattributed to the novel beta-coronavirus. As human-to-human transmissionof the virus is not sustained, a large zoonotic reservoir may serve as aprincipal source for transmission events. The high case fatality rate,vaguely defined epidemiology, and absence of prophylactic or therapeuticmeasures against this novel virus have created an urgent need for aneffective vaccine and related therapeutic agents.

SUMMARY

Disclosed herein are new methods for inducing an immune response toMERS-CoV spike (S) protein that are surprisingly effective for inducingneutralizing antibody responses to MERS-CoV in a subject. The methodsare useful, for example, for preventing or treating a MERS-CoV infectionin the subject.

In several embodiments, the method includes administering a prime-boostvaccination to the subject, comprising administering a nucleic acidmolecule encoding a MERS-CoV S protein, and polypeptide comprising orconsisting of a S1 subunit of the MERS-CoV S protein (MERS-CoV S1protein), to the subject to generate the immune response to the MERS-CoVS protein. In a non-limiting example, the prime-boost vaccination cancomprise a prime comprising administering the nucleic acid moleculeencoding the MERS-CoV S protein to the subject, a first boost,comprising administering a therapeutically effective amount of thenucleic acid molecule encoding the MERS-CoV S protein to the subject,and a second boost comprising administering a therapeutically effectiveamount of a MERS-CoV S1 protein to the subject. In some embodiments, theMERS-CoV S protein can comprise or consist of the amino acid sequenceset forth as SEQ ID NO: 14, and/or the MERS-CoV S1 protein can compriseor consist of the amino acid sequence set forth as SEQ ID NO: 16.

Additionally, novel immunogens including the MERS-CoV S protein or afragment thereof (such as the receptor binding domain, RBD) areprovided. In some embodiments, a polypeptide including the RBD ofMERS-CoV S protein linked to a protein nanoparticle is provided, whichcan be used to generate protein nanoparticles that display the MERS-CoVS protein RBD. Nucleic acid molecules encoding the polypeptides are alsoprovided.

The disclosure also provides isolated monoclonal antibodies and antigenbinding fragments that specifically bind to an epitope on MERS-CoV Sprotein. Also disclosed are compositions including the antibodies andantigen binding fragments, nucleic acids encoding the antibodies andantigen binding fragments, expression vectors comprising the nucleicacids, and isolated host cells that express the nucleic acids. Theantibodies and antigen binding fragments can neutralize MERS-CoVinfection, and therefore can be used in methods of treating orpreventing a MERS-CoV infection in a subject. The methods includeadministering a therapeutically effective amount of one or more of theantibodies, antigen binding fragments, nucleic acid molecules, vectors,or compositions disclosed herein, to the subject, for example to asubject at risk of or having MERS-CoV infection.

The foregoing and other features and advantages of this disclosure willbecome more apparent from the following detailed description of severalembodiments which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C illustrate MERS-CoV Spike glycoprotein vaccine design andimmunogenicity in mice. Candidate vaccine immunogens were designedaround the sole surface glycoprotein of beta-cornaviruses. (A) Schematicrepresentation of MERS-CoV S protein cDNAs and recombinant proteins.Five vaccine constructs were made: three DNA and two protein subunits.DNA constructs consisted of full-length S or truncated versions thateither had the transmembrane domain or the entire S2 subunit deleted.The protein constructs contain either a truncated S molecule with thetransmembrane domain deleted (S-ΔTM) or the S1 subunit. RBD: receptorbinding domain; SP: signal peptide, TM: transmembrane domain; FTH:Foldon (trimerization domain), Thrombin (cleavage site) followed byhistidine tag; 3CHis: Human rhinovirus 3C protease cleavage site,followed by 6× histidine tag. (B) Immunogenicity of eight vaccineregimens. Five mice per group were immunized with plasmid DNA only atweeks 0, 3 and 6 (groups 1-3); plasmid DNA at weeks 0 and 3 and proteinplus Ribi adjuvant at week 6 (groups 4-6); or protein plus Ribi adjuvantat weeks 0 and 4 (groups 7, 8). Two weeks after each immunization,neutralizing antibody titers were measured against pseudotyped MERS-CoVEngland1 virus. Open, grey and black bars respectively represent theIC₉₀ neutralization titers (GMT with 95% CI) from the post-prime, firstpost-boost, and second post-boost sera. A non-parametric two-tailedt-test (Mann-Whitney) was used for statistical analysis, and therelevant P values are indicated. (C) MERS-CoV vaccines inducedcross-neutralization to eight MERS-CoV strains. The sera from the miceimmunized with MERS-CoV S DNA three times, primed with S DNA and boostedwith S1 protein plus Ribi adjuvant, or primed and boosted with S1protein plus Ribi adjuvant were assayed for neutralization to the eightstrains of MERS-CoV and SARS-CoV pseudotyped viruses as indicated. IC₉₀titer is shown. Data are presented as the mean of triplicates withstandard errors.

FIGS. 2A and 2B illustrate the antisera targets of neutralization.Immunization with different vaccine regimens elicited neutralizingantibodies that target the Spike (S) glycoprotein within and outside thereceptor-binding domain (RBD). (A) Cell adsorption assay. Sera from miceimmunized with MERS-CoV S DNA only, S DNA prime and S1 protein plus Ribiadjuvant, or S1 protein plus Ribi adjuvant prime and boost wereevaluated for neutralization activity against pseudotyped MERS-CoV(Eng1) after adsorption with 293T cell surface-expressed MERS-CoV Spikeproteins: S, RBD, S1, S2. Serum neutralization was tested at a singledilution. Sera adsorbed with untransfected 293T cells served as controlsand retained 95% of neutralization activity. Each bar represents themean of triplicate assays with standard errors. (B) Protein competitionneutralization assay. Sera at a single dilution from the immunized micewere also assayed for neutralization of MERS-CoV England1 pseudovirus inthe presence of soluble MERS-CoV RBD, S1 and S2 proteins atconcentrations of 0.016 to 50 μg/ml.

FIGS. 3A and 3B illustrate monoclonal antibody (mAb) binding andneutralization activity. Four mAbs were characterized for their bindingspecificity and neutralizing activity. (A) Binding specificity. Each ofthe mAbs was tested, by ELISA, for binding to soluble receptor bindingdomain (RBD), S1, and S2 conjugated to Fc for stabilization (S2-hFc).(B) Binding affinity and neutralization. Binding affinity andneutralization activity were measured by biolayer interferometry (rawdata shown in FIG. 19) and a pseudotyped MERS-CoV (England1) virusneutralization assay, respectively. The mAbs specific for the MERS-CoVRBD—D12 and F11—demonstrated the highest neutralization potency of thefour characterized mAbs. G2, which bound S1 outside the RBD, had weakeraffinity to S1 than D12 and F11 but near equal neutralization potency.The S2-binding mAb, G4, had a tenfold lower neutralizing potency thanthe other mAbs.

FIGS. 4A-4E illustrate the molecular characterization of MERS-CoVneutralizing mAbs. Vaccine-induced mAb D12 binds directly to the DPP4interacting region of the MERS-CoV Spike receptor binding domain (RBD)and effect neutralization by directly blocking receptor binding. (A)(left) Comparison of RBD binding to D12 antibody and RBD binding byDPP4. RBD with receptor binding motif (RBM, residues 484-567, magenta)and D12 are shown in cartoon representation. The main interactingregions are contained within CDR H2, CDR H3, and CDR L2. (right) DPP4 isshown in cartoon format with Asparagine 229 and attached N-glycan shownin stick representation. The RBD molecule is oriented identical to(left). (B) Interfaces for antibody:RBD and DPP4:RBD crystal structurecomplexes. (left) RBD in surface representation is shown with the D12heavy chain and light chain paratopes, respectively. The CDR loops areshown in ribbon representation. (center) The RBD is rotated to show thefull D12 paratope with D12 CDR H2 interacting with the RBD W535 and E536residues that predominantly interact with the Asparagine 229 associatedN-glycan on DPP4. (right) RBD is shown in surface representation withthe DPP4 interacting region. Major interacting regions from DPP4 areshown in cartoon representation with asparagine 229 and N-glycan alsoshown in stick representation in the same way as is shown for the SARSmAbs (FIG. 16). (C) Crystal structure of MERS-CoV England1 RBD andeffect of critical RBD mutations on binding. (D) D12 and RBD interface.All CDRs are shown in cartoon format and interacting residues are shownin stick representation with hydrogen bonds depicted by dotted lines.(D, E) RBD residues 506 and 509 that have been observed with variousmutations in isolated viruses are highlighted in green. Critical RBDresidues identified by structural definition and viral resistanceevolution 532, 535 and 536 that reduce or eliminate D12 binding arehighlighted. ELISA results show that these mutations can effectivelyeliminate F11 or D12 binding.

FIGS. 5A-5D illustrate MERS-CoV Spike glycoprotein vaccine design,immunogenicity, and efficacy in non-human primates. Selected candidatevaccine immunogens based on mice studies were evaluated in non-humanprimates (NHP). (a) Schematic representation of full-length MERS-CoVSpike protein cDNA and recombinant S1 protein. Two vaccine constructswere tested: one DNA and one protein subunit. DNA construct consisted offull-length S that had the transmembrane domain. The protein constructcontains a truncated S molecule with the S1 subunit. RBD: receptorbinding domain; SP: signal peptide, TM: transmembrane domain; 3CHis:Human rhinovirus 3C protease cleavage site, followed by 6× histidinetag. (b) Immunogenicity of three vaccine regimens. Six NHP per groupwere immunized intramuscularly with plasmid DNA only, followed byelectroporation, at weeks 0, 4 and 8; plasmid DNA and electroporation atweeks 0 and 4; and protein plus aluminum phosphate at week 8 or proteinplus aluminum phosphate at weeks 0 and 8. Two weeks after eachimmunization and at week 12 and week 18, neutralizing antibody titerswere measured against pseudotyped MERS-CoV England1 virus. Differentsymbols indicate sera from 6 NHPs per group that were collected atindicated time points. IC₉₀ neutralization titers (GMT with 95% CI) fromthe sera were determined. Each data point represents the mean oftriplicate assays. Assays were repeated once. A non-parametrictwo-tailed t-test (Mann-Whitney) was used for statistical analysis, andthe relevant P values are indicated. (c) MERS-CoV Spike glycoproteinimmunogens protect against pulmonary disease in non-human primates(NHPs). Six unimmunized NHPs and 12 NHPs that were immunized with one oftwo selected candidate vaccine immunogens (1. full-length S DNA prime/S1subunit protein boost; 2. S1 subunit protein prime/S1 subunit proteinboost) were challenged with MERS-CoV 19 weeks after last vaccine boost.Each NHP was intra-tracheally administered 5×10⁶ PFU of the Jordan N3strain of MERS-CoV (GenBank ID: KC776174.1). The percent abnormal lungvolume in all NHPs peaked on day 3 post-challenge; however, the lunginfiltrates were significantly more extensive and prolonged in theunvaccinated compared to vaccinated NHPs. A non-parametric two-tailedt-test (Mann-Whitney) was used for statistical analysis. One star (*)represents P values less than 0.05, two stars (**) indicate P valuesless than 0.01. (d) Abnormal lung segmental images from selected animalson day 6 post challenge are shown. The images correspond to data pointscircled in black in FIG. 5C. The CT images and abnormal lung segmentalimages for all 18 animals are shown in FIGS. 18A-18C.

FIGS. 6A-6D illustrate that MERS-CoV pseudovirus utilizes human DPP4 totransduce target cells. (A) DPP4 expression on the cell surface. Huh7.5cells (left panel), DPP4-untransfected 293 cells (middle panel), andDPP4-transfected 293 cells (right panel) were stained with goatanti-DPP4 antibody and control antibody and analyzed by flow cytometry.(B) Transduction of DPP4-expressing cells by pseudotyped MERS-CoVEngland1 virus. Huh7.5 and 293 cells without and with DPP4-transfection(in grey and black bars) were transduced by MERS-CoV pseudotyped virus.Relative expression of luciferase activity was measured (CPS).Untransfected and untransduced cells were used as the background control(open bars). (C) Transduction of Huh7.5 cells by MERS-CoV pseudoviruswas blocked by soluble human DPP4 (sDPP4). MERS-CoV England1 pseudoviruswas incubated with soluble human DPP4 before transduction of Huh7.5cells. Relative luciferase activity (CPS) is shown. (D) Transduction ofHuh7.5 cells by MERS-CoV pseudovirus was blocked by anti-DPP4, but notanti-ACE2 antibody. Huh7.5 cells were incubated with anti-DPP4 oranti-ACE2 polyclonal antibodies and then transduced with MERS-CoVEngland1 pseudovirus. Relative luciferase activity (CPS) is shown.

FIG. 7 illustrates a comparison of MERS-CoV Spike (S) glycoproteinsequences across strains used for a pseudotyped virus neutralizationassay panel. A schematic representation of MERS-CoV S protein is shownwith the N-terminal domain (NTD), receptor binding domain (RBD), heptadrepeats 1 and 2 (HR1 and HR2), and transmembrane domain (TM). EightMERS-CoV S sequences published in GenBank were aligned with the England1strain. Several amino acid differences are shown with the England1strain as the referent. Phylogenetic distance between strains isrepresented by branch length on the phylogenetic tree to the left of thesequences.

FIG. 8 illustrates that MERS-CoV vaccine elicited virus neutralizationresponses as measured by both pseudotyped and live virus neutralizationassays. Eight groups of mice were immunized as indicated in MG. 1.Neutralizing antibodies from sera five weeks after last vaccine boostwere measured by a pseudovirus neutralization assay (black bars) andlive virus micro-neutralization assay (open bars) to MERS-CoV JordanN3respectively.

FIGS. 9A-9C illustrate that MERS-CoV S DNA immunization induced antibodybinding to both S1 and S2. (A and B) Schematic representation ofMERS-CoV Spike DNA and protein constructs used for cell adsorptionassays. (C) Sera from mice immunized with MERS-CoV S DNA, primed with SDNA and boosted with S1 protein plus Ribi adjuvant, or primed andboosted with S1 protein plus Ribi adjuvant were assayed by flowcytometry for their binding to cell surface-expressed MERS-CoV Spikeproteins. 293T cells transfected with MERS-CoV S, RBD-HA™, S1-TM andS2-TM were incubated with sera from the three immunization groups (1:200dilution) and then stained with anti-mouse PE conjugate. S: Spikeglycoprotein, RBD: receptor binding domain, HA: hemagglutinin, TM:transmembrane.

FIGS. 10A and 10B illustrate that MERS-CoV S DNA/S1 protein prime-boostvaccination in mice induced a Th1-biased IgG response compared to aTh2-biased response elicited by a S1 protein prime-boost regimen. (A)Sera from mice immunized with MERS-CoV S DNA, primed with S DNA andboosted with S1 protein plus Ribi adjuvant, or primed and boosted withS1 protein plus Ribi adjuvant were assayed, by ELISA, for theirpredominance of MERS-CoV S1-specific IgG1 and IgG2a antibody responses.Open and black circles represent IgG2a and IgG1 antibody titers(Geometric mean titer (GMT) with 95% CI), respectively. (B) The GMTratios of IgG2a to IgG1 in the three groups were calculated from theleft panel.

FIG. 11 illustrates identification of monoclonal antibodies against theMERS-CoV Spike glycoprotein. Immunized mice in both DNA and proteinvaccine groups had their spleens harvested three days after anadditional S1 protein. Splenocytes were then fused with Sp2/0 myelomacells to generate hybridomas that underwent three rounds of screeningfor binding to the S1, RBD, and S2 domains. The final round of screensgenerated 45 subclones. Supernatant from the subclones culture weresubjected to neutralization and binding tests. Percentage ofneutralization against pseudoviruses of Eng1 strain from the subcloneswas determined and is shown. Supernatants from the subclones wereassessed for binding to the RBDs, S1 and S-ΔTM proteins. Western blotanalysis was done to assess whether the subclones can recognizedenatured S linear eptitopes. Based on the ELISA binding data, the mAbswere classified into three groups: RBD specific, S1 specific (non-RBD)and S2 specific as indicated. Four of these mAbs (D12, F11, G2 and G4)were selected for additional characterization based on their antigenicspecificity and high neutralization potency.

FIG. 12 shows Octet Biosensorgrams of MERS-CoV S1, MERS-CoV RBD, andMERS-CoV S2 molecules binding to vaccine-induced mouse monoclonal IgGs.Mouse monoclonal antibodies were loaded onto AMC probes and associationwith MERS-CoV antigen was allowed to proceed for 300 s, followed bydissociation for 300 s with the responses measured in nm using an OctetRed 384 machine. The S2 binding to G4 was measured by loadinghuman-Fc-S2 onto AHC probes and measuring association with varyingconcentrations of G4 Fab. The solid black lines represent the best fitof the kinetic data to a 1:1 binding model. All experiments were carriedout at 30° C. in PBS buffer (pH 7.4) supplemented with 1% BSA tominimize non-specific binding. The dotted line indicates the beginningof dissociation and the legend indicates the MERS-CoV antigen and G4 Fabconcentrations used.

FIG. 13 shows immunofluorescence of monoclonal antibodies (mAb) specificfor MERS-CoV infected cells. Red Alexa Fluors 546 signal for isolatedmAbs D12, F11, G2 and G4 binding to MERS-CoV (EMC strain)-infected Verocells was robust for serial mAb dilutions down to 0.00125 μg/μL. Purpleregions indicated the nucleus of the Vero cells and 0 μg/ml mAbconcentration was used as the control.

FIGS. 14A-14C show the specificity of the MERS-CoV Spike glycoproteinmAbs. (A) Protein competition neutralization assay. mAbs at a singledilution were assayed for neutralization of MERS-CoV England1pseudovirus in the presence of soluble MERS-CoV RBD, S1 and S2 proteinsat concentrations of 0.016 to 50 μg/ml. (B, C) mAbs were assayed forneutralization to the MERS-CoV or mutant pseudotyped viruses. Data arepresented as the mean of triplicates with standard errors. Onerepresentative of two repeated experiments is shown.

FIGS. 15A-15C illustrate the assessment of D12 and F11 interactions withMERS-CoV RBD. (A) D12 and F11 directly block RBD binding to DPP4. Mousemonoclonal antibodies D12 and F11 were loaded onto AMC probes for 300 sand association with MERS-CoV RBD was allowed to proceed for 300 s,followed by incubation with soluble DPP4 for 300 s with the responsesmeasured in nm using an Octet Red 384 machine. (B) Multiple neutralizingepitopes are accessible on MERS CoV RBD. MERS CoV RBD was loaded ontoanti-penta-His probes for 300 s followed by sequential binding of bothD12 and F11 mAbs. (C) Soluble DPP4 prevents binding of RBD to D12 andF11 mAbs. MERS CoV RBD was loaded onto anti-penta-His probes for 300 sfollowed by sequential binding of DPP4 and either D12 or F11 mAbs. Allexperiments were carried out at 30° C. in PBS buffer (pH 7.4)supplemented with 1% BSA to minimize non-specific binding. The dottedline indicates the beginning of incubation with the second and thirdligand in each of the experiments.

FIGS. 16A-16D show published structures of SARS-CoV neutralizingantibodies that effectively block the receptor interacting region of thevirus. (A) SARS receptor binding domain (RBD) with the receptor bindingmotif (RBM) and the ACE2 receptor are shown in cartoon representation.The ACE2 interacting region is mapped onto the SARS RBD (surfacerepresentation, rotated). The RBD region that interacts with the ACE2glycan is shown. (B-D) SARS RBD and M396 Fab (Prabakaran et al., J BiolChem 281, 15829, 2006; Zhu et al., PNAS 104, 12123, 2007), F26G19 Fab(Pak et al., J mol biol 388, 815, 2009) and 80R Fv (Hwang et al., J biolchem 281, 34610, 2006) are shown in cartoon representation. The antibodyinteracting region is mapped onto the RBD.

FIG. 17 shows sera from vaccinated non-human primates (NHPs) blocked thebinding of murine monoclonal antibodies to MERS-CoV Spike protein.Serial dilutions of mixed NHP sera from three vaccinated groups weretested in competition with biotinylated monoclonal antibodies F11, D12,G2, G4 for binding to MERS-CoV S1 or S-dTM (for mAb G4). Percentinhibition is shown.

FIGS. 18A-18C show three dimensional computed tomography (CT)visualizations of lungs from non-human primates (NHPs) challenged withMERS-CoV. Unvaccinated NHPs (A) and those vaccinated with S DNA/S1protein (B) or S1 protein/S1 protein (C) underwent chest CT imagingbefore virus challenge and days 3, 6, 9, and 14 post-challenge. Twodimensional coronal CT images and three dimensional reconstructionsshowed larger volumes of percent abnormal lung (infiltrate,consolidation, ground glass opacity) in the unvaccinated compared tovaccinated NHPs.

FIGS. 19A-19D illustrate how non-human primate (NHP) anti-MERS-CoVantibody responses increase post-challenge but do not correlate withpulmonary disease. (A) ELISA IgG antibody titers and (B) neutralizationtiters both rise after challenge with MERS-CoV. There was no significantcorrelation between neutralization titers of NHP sera at day ofchallenge (C) or at peak (2 weeks after last boost) (D) and lung diseaseas measured by percent abnormal lung volume on computed tomography.Graphpad Prism 6 was used to determine the Pearson correlationcoefficients and corresponding p values.

FIG. 20 is a table showing crystallographic data collection andrefinement statistics for the crystal structure of D12 Fab with theMERS-CoV RBD (England1 strain).

FIGS. 21A-21C are a set of tables showing D12 antibody interactions withMERS-CoV S protein RBD (England1 strain) as determined from the crystalstructure of D12 Fab with the RBD.

FIG. 22 is a graph and a schematic diagram indicating the structure ofMERS-CoV S protein, and the specificity (RBD, S1 (non-RBD), or S2) andsource of identified S protein specific antibodies.

FIG. 23 shows a set of graphs illustrating the neutralization activityof identified NHP antibodies (JC57-11, JC57-14, JC57-13, FIB_B2, andFIB_H1), human antibodies (C2, C5, A2, and A10), and murine antibodies(D12, F11, G2 and G4). Neutralization activity was assayed using apseudovirus neutralization assay for the MERS-CoV EMC strain asdescribed in the examples.

FIG. 24 shows a set of graphs illustrating the neutralization activityof the JC57-11, JC57-14, C2, and C5 antibodies. Neutralization activitywas assayed using a pseudovirus neutralization assay for the indicatedstrains of MRES-CoV.

FIG. 25 shows a set of graphs illustrating the neutralization activityof the A2, JC57-13, A10, and G2 antibodies. Neutralization activity wasassayed using a pseudovirus neutralization assay for the indicatedstrains of MRES-CoV.

SEQUENCES

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. The Sequence Listing is submitted as an ASCII textfile in the form of the file named “Sequence.txt” (˜164 kb), which wascreated on May 3, 2019, and which is incorporated by reference herein.In the accompanying sequence listing:

SEQ ID NO: 1 is an exemplary nucleotide sequence encoding the V_(H) ofthe JC57-13 non-human primate (NHP) antibody (VRC 4230).caggtgcagctgcaggagtcgggcccaggactggtgaagccttcggagaccctgtccctcacctgcgccgtctctggtggctccatcagcagtaactactggaactggatccgccagtccccagggaaggggctagagtggattgggtatatctatggtggtagtgggagcaccacctacaacccctccctcaagagtcgagtcgccatttcaacagacacgtccaaggaccagttttccctgaagctgagctctgtgaccgccgcggacaccgccgtatattactgtgcgagactgctgcccttaggggggggatactgctttgactactggggccagggagtcctggt caccgtctcctca SEQ IDNO: 2 is the amino acid sequence of the V_(H) of the JC57-13 NHPantibody (VRC 4230). QVQLQESGPGLVKPSETLSLTCAVSGGSISSNYWNWIRQSPGKGLEWIGYIYGGSGSTTYNPSLKSRVAISTDTSKDQFSLKLSSVTAADTAVYYCARLL PLGGGYCFDYWGQGVLVTVSSSEQ ID NO: 3 is an exemplary nucleotide sequence encoding the V_(L) ofthe JC57-13 NHP antibody (VRC 4231).Gatattgtgatgacccagactccattcaccctgcccgtcacccctggagaggcggcctccatctcctgcaggtctagtcagagcctcttcgatagtgattatggaaacacctatttggattggtatctgcagaagccaggccagtctccacagctcctgatctatatgctttccaaccgggcctctggagtccctgataggttcagtggcagtgggtcaggcactgatttcacactgaaaatcagccgggtggaggctgaggatgttgggttatattactgcatgcaaagtgtagagtatccattcactttcggccccgggaccaaactggatatcaaa SEQ ID NO: 4 is the amino acidsequence of the V_(L) of the JC57-13 NHP antibody (VRC 4231).DIVMTQTPFTLPVTPGEAASISCRSSQSLFDSDYGNTYLDWYLQKPGQSPQLLIYMLSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGLYYCMQSVEY PFTFGPGTKLDIK SEQ IDNO: 5 is an exemplary nucleotide sequence encoding the V_(H) of theJC57-11 NHP antibody (VRC 4232).Gaggtgcagctgctggagtcgggcccaggagtggtgaggccttcggagaccctgtccctctcctgcgctgtctctggtggctccatcagcgatagttaccggtggagctggatccgccagcccccagggaagggactggagtgggttggctacatctttgctactggtacgaccaccaactacaacccctccctcaagagtcgagtcaccatttcaaaagacacgtccaagaaccagttctccttgaagctgagctctgtgaccgccgcggacacggccgtttactactgtgcgagagagccgttcaaatattgtagtggtggtgtctgctatgcccacaaggacaactcattggatgtctggggccagggagttctggtcaccgtctcctca SEQ ID NO: 6 is the aminoacid sequence of the V_(H) of the JC57-11 NHP antibody (VRC 4232).EVQLLESGPGVVRPSETLSLSCAVSGGSISDSYRWSWIRQPPGKGLEWVGYIFATGTTTNYNPSLKSRVTISKDTSKNQFSLKLSSVTAADTAVYYCAREPFKYCSGGVCYAHKDNSLDVWGQGVLVTVSS SEQ ID NO: 7 is an exemplary nucleotidesequence encoding the V_(L) of the JC57-11 NHP antibody (VRC 4233).Gaaattgtgatgacgcagtctccagccaccctgtctttgtctccaggggaaagagccactctctcctgcagggccagtcagagtgttagtagcaacttagcctggtaccagcagaaacctgggcaggctcccaggctcctcatccacagtgcgtccagcagggccactggcatcccagacaggttcagtggcagcgggtctgggacagagttcagtctcaccatcagcagtctggaggctgaagatgttggagtttatcactgctatcagcatagcagcgggtacactttcggccccggg accaaactggatatcaaaSEQ ID NO: 8 is the amino acid sequence of the V_(L) of the JC57-11 NHPantibody (VRC 4233). EIVMTQSPATLSLSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIHSASSRATGIPDRFSGSGSGTEFSLTISSLEAEDVGVYHCYQHSSGYTFGPG TKLDIK SEQ ID NO: 9is an exemplary nucleotide sequence encoding the V_(H) of the JC57-14NHP antibody (VRC 4234).Gaggtgcagctgcaggagtcgggcccaggactggtgaagccttcggagaccctgtccctcacctgcgctgtctctggtgactccatcagcagtaactactggagctggatccgccagcccccagggaagggactggagtggattggacgtttctctggtagtggtgggagcaccgacttcaacccctccctcaagagtcgggtcaccatttcaacagacacgtccaagaaccagttctccctgaacctgaggtctgtgaccgccgcggacacggccgtgtattactgtgcgaaaacctatagcggcacctttgactactggggccagggagtcctggtcaccgtctcctc a SEQ ID NO: 10 isthe amino acid sequence of the V_(H) of the JC57-14 NHP antibody (VRC4234). QVQLQESGPGLVKPSETLSLTCAVSGDSISSNYWSWIRQPPGKGLEWIGRFSGSGGSTDFNPSLKSRVTISTDTSKNQFSLNLRSVTAADTAVYYCAKTY SGTFDYWGQGVLVTVSS SEQID NO: 11 is an exemplary nucleotide sequence encoding the V_(L) of theJC57-14 NHP antibody (VRC 4235).Gacattcagatgacgcagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcgagtcaggacattaacaattatttaagttggtatcagcagaaaccagggaaagcccctaagcccctgatctattatgcatccagtttggaaacaggagtaccttcaaggttcagtggaagtagatctgggacagattacactctcaccatcagcagtctgcagcttgaagattttgcaacatattactgtcaacagtataataattccccgtacagttttggccag gggaccaaagtggagatcaaaSEQ ID NO: 12 is the amino acid sequence of the V_(L) of the JC57-14 NHPantibody (VRC 4235). DIQMTQSPSSLSASVGDRVTITCRASQDINNYLSWYQQKPGKAPKPLIYYASSLETGVPSRFSGSRSGTDYTLTISSLQLEDFATYYCQQYNNSPYSFGQ GTKVEIK

SEQ ID NOs: 13 and 14 are nucleic acid and protein sequences of thefull-length MERS-CoV S protein, England1 strain.

SEQ ID NOs: 15 and 16 are nucleic acid and protein sequences of the S1subunit of the MERS-CoV S protein, England1 strain.

SEQ ID NOs: 17 and 18 are nucleic acid and protein sequences of afragment of the MERS-CoV S protein, England1 strain, including thereceptor binding domain (RBD).

SEQ ID NOs: 19 and 20 are nucleic acid and protein sequences of theS-ΔTM fragment of the MERS-CoV S protein, England1 strain.

SEQ ID NO: 21 is the amino acid sequence of a ferritin nanoparticlesubunit.

SEQ ID NOs: 22 and 23 are amino acid sequences of MERS-CoV S protein RBDdomains linked to a ferritin nanoparticle subunit.

SEQ ID NOs: 24 and 25 are the amino acid sequences of signal peptides.

SEQ ID NOs: 26 and 27 are polynucleotide sequences encoding MERS-CoV Sprotein RBD domains linked to a ferritin nanoparticle subunit.

SEQ ID NO: 28 is the amino acid sequence of a lumazine synthasenanoparticle subunit.

SEQ ID NO: 29 is the amino acid sequence of an encapsulin nanoparticlesubunit.

SEQ ID NOs: 30 and 31 are amino acid sequences of MERS-CoV S protein RBDdomains linked to an encapsulin nanoparticle subunit.

SEQ ID NOs: 32 and 33 are polynucleotide sequences encoding MERS-CoV Sprotein RBD domains linked to an encapsulin nanoparticle subunit.

SEQ ID NO: 34 is the amino acid sequence of a Sulfur Oxygenase Reductasenanoparticle subunit.

SEQ ID NO: 35 is an exemplary nucleotide sequence encoding the V_(H) ofthe C2 human antibody (VRC 4792).Caggtgcagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgcaaggcttctggaggcaccttcagcatctatgctatcagctgggtgcgacaggcccctggacaagggcttgagtggatgggagggatcatccctatctttggtacagcaaactacgcacagaagttccagggcagagtcacgattaccgcggacaaatccacgagcacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcgagagaggggggccaccagggatattgtagtggtggtagctgctacgactttgactactggggccagggaaccctggtcaccgtctcctca SEQ ID NO: 36 is the amino acid sequence of the V_(H) of theC2 human antibody (VRC 4792)QVQLVQSGAEVKKPGSSVKVSCKASGGTFSIYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREGGHQGYCSGGSCYDFDYWGQGTLVTVSS SEQ ID NO: 37 isan exemplary nucleotide sequence encoding the V_(L) of the C2 humanantibody (VRC 4793).gatgttgtgatgactcagtctccactctccctgcccgtcacccctggagagccggcctccatctcctgcaggtctagtcagagcctcctgcatagtaatggatacaactatttggattggtacctgcagaagccagggcagtctccacagctcctgatctatttgggttctaatcgggcctccggggtccctgacaggttcagtggcagtggatcaggcacagattttacactgaaaatcagcagagtggaggctgaggatgttggggtttattattgcatgcaagctctacaaactcctgcgttcggcggagggaccaagctggagatcaaa SEQ ID NO: 38 is the amino acidsequence of the V_(L) of the C2 human antibody (VRC 4793).DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPAFGGGTKLEIK SEQ ID NO: 39 is an exemplarynucleotide sequence encoding the V_(H) of the C5 human antibody (VRC4794).cagctgcagctgcaggagtcgggcccaggactggtgaagccttcggagaccctgtccctcacctgcactgtctctggtggctccatcagcagtagtagttactactggggctggatccgccagcccccagggaaggggctggagtggattgggagtatctattatagtgggagcacctactacaacccgtccctcaagagtcgagtcaccatatccgtagacacgtccaagaaccagttctccctgaagctgagctctgtgaccgccgcagacacggctgtgtattactgtgcgagcctcttaaggcccctgatttattgtagtggtggtagctgcaccgactactggggccagggaaccctggtcaccgtctcctca SEQ ID NO: 40 is the amino acid sequence of the V_(H) ofthe C5 human antibody (VRC 4794).QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASLLRPLIYCSGGSCTDYWGQGTLVTVSS SEQ ID NO: 41 isan exemplary nucleotide sequence encoding the V_(L) of the C5 humanantibody (VRC 4795).Cagtctgccctgactcagcctgcctccgtgtctgggtctcctggacagtcgatcaccatctcctgcactggaaccagcagtgacgttggtggttataactatgtctcctggtgccaacagcacccaggcaaagcccccaaactcatgatttatgaggtcagtaatcggccctcaggggtttctaatcgcttctctggctccaagtctggcaacacggcctccctgaccatctctgggctccaggctgaggacgaggctgattattactgcagctcatatacaagcaacatcactcttgtcttcggaactgggaccaaggtcaccgtccta SEQ ID NO: 42 is the amino acidsequence of the V_(L) of the C5 human antibody (VRC 4795).QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWCQQHPGKAPKLMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSNITLVFGTGTKVTVL SEQ ID NO: 43 is an exemplarynucleotide sequence encoding the V_(H) of the A2 human antibody (VRC4796).caggtgcagctggtggagtctgggggaggcttggtcaagcctggagggtccctgagactctcctgtgcagcctctggattcaccttcagtgactactacatgagctggatccgccaggctccagggaaggggctggagtgggtttcatacattagtagtagtggtagtaccatatactacgcagactctgtgaagggccgattcaccatctccagggacaacgccaagaactcactgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgagagtagggttaggcagtggctggtacgactggttcgacccctggggccagggaaccctggtcaccgtctcctca SEQ ID NO: 44 is the amino acid sequence of the V_(H) of the A2 humanantibody (VRC 4796).QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVGLGSGWYDWFDPWGQGTLVTVSS SEQ ID NO: 45 is anexemplary nucleotide sequence encoding the V_(L) of the A2 humanantibody (VRC 4797).cagtctgccctgactcagccgccctcagtgtctggggccccagggcagagggtcaccatctcctgcactgggagcagctccaacatcggggcaagttatgatgtacactggtaccagcaccttccaggaacagcccccaaactcctcatctatggtaacaccaatcggccctcaggggtccctgaccgattctctggctccaagtctggcacctcagcctccctggccatcactgggctccaggctgaggatgaggctgattattactgccagtcctatgacagcagcctgagtggtgtggtattcagcggagggaccaagctgaccgtcctag SEQ ID NO: 46 is the aminoacid sequence of the V_(L) of the A2 human antibody (VRC 4797).QSALTQPPSVSGAPGQRVTISCTGSSSNIGASYDVHWYQHLPGTAPKLLIYGNTNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGVVFSGGTKLTVL SEQ ID NO: 47 is an exemplarynucleotide sequence encoding the V_(H) of the A10 human antibody (VRC4798).caggtgcagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgcaaggcttctggaggcaccttcagcacctatgctctcagctgggtgcgacaggcccctggacaagggcttgagtggatgggagggatcatccctatctttggtacagcaaactacgcacagaagttccagggcagagtcacgattaccgcggacgaatccacgagcacggcctacatggagttgaacagcctgagatctgaggacacggccgtgtattactgtgcgagaggaagccggagcagctcttccgctgaatacttccagcactggggccagggcaccctggtcaccgtctcctca SEQ ID NO: 48 is the amino acid sequence of the V_(H) of the A10 humanantibody (VRC 4798).QVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYALSWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELNSLRSEDTAVYYCARGSRSSSSAEYFQHWGQGTLVTVSS SEQ ID NO: 49 is anexemplary nucleotide sequence encoding the V_(L) of the A10 humanantibody (VRC 4799).cagtctgccctgactcagcctcgctcagtgtccgggtctcctggacagtcagtcaccatctcctgcactggaaccagcagtgatgttggtggttataactatgtctcctggtaccaacagcacccaggcaaagcccccaaactcatgatttatgatgtcagtaagcggccctcaggggtccctgatcgcttctctggctccaagtctggcaacacggcctccctgaccatctctgggctccaggctgaggatgaggctgattattactgctgctcatatgcaggcagctacactttagaagtggtattcggcggagggaccaagctgaccgtcctag SEQ ID NO: 50 is the aminoacid sequence of the V_(L) of the A10 human antibody (VRC 4799).QSALTQPRSVSGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSKRPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCCSYAGSYTLEVVFGGGTKLTVL SEQ ID NO: 51 is an exemplarynucleotide sequence encoding the V_(H) of the FIB_B2 NHP antibody (VRC5069).caggtgcagctgcaggagtcgggcccaggactggtgaagccttcggagaccctgtctctcacctgcgctgtttctggtggctccatcagcagcaactactggtactggatccgccagtccccagtgaaggggctggagtggattgggtatatctatggtggtagtgggggcaccgaatacaacccctccctcaagagtcgagtcaccatttcaacagacacgtccaagaaccagtttttcctgaagctgagctctgtgaccgccgcggacaccgccgtatattactgtgcgagatccttttatagctggaacggggaatcctggggccaaggggtcgtcgtcaccgtctcctca SEQ IDNO: 52 is the amino acid sequence of the V_(H) of the FIB_B2 NHPantibody (VRC 5069).QVQLQESGPGLVKPSETLSLTCAVSGGSISSNYWYWIRQSPVKGLEWIGYIYGGSGGTEYNPSLKSRVTISTDTSKNQFFLKLSSVTAADTAVYYCARSFYSWNGESWGQGVVVTVSS SEQ ID NO: 53 is anexemplary nucleotide sequence encoding the V_(L) of the FIB_B2 NHPantibody (VRC 5070).gacattcagatgtcccagactccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagggcattaacgattatttaaattggtatcagcagaaaccggggaaagcccctaagctcctgatctattatggaaacagtttggcaagtggggtcccatcaaggttcagtggcagtggttctgggacagatttctctctcaccatcagcagcctgcagcctgaagattttgcaacttattactgtcaacagggtgatagtttccctctcactttcggcggagggaccaaagtggatatcaaa SEQ ID NO: 54 is the amino acid sequenceof the V_(L) of the FIB_B2 NHP antibody (VRC 5070).DIQMSQTPSSLSASVGDRVTITCRASQGINDYLNWYQQKPGKAPKLLIYYGNSLASGVPSRFSGSGSGTDFSLTISSLQPEDFATYYCQQGDSFPLTFGGGTKVDIK SEQ ID NO: 55 is an exemplarynucleotide sequence encoding the V_(H) of the FIB_H1 NHP antibody (VRC5071).gaggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaagcttctggacacattttcaccagttatgttatcaactggctgcaagaggcccctggacaagggtttgagtggatgggaggaatccaccctggtaatggtggcagagactacgcacagaagttccagggcagagtcacgattaccgcggacatgtccacgagcacagtctacatggagctgagaagtctgagatctgaggacatggccgtgtattactgtgcagcatccagtggtagttatggtgttagctcattggatgtctggggccggggagttctggtcaccgtctcctcaSEQ ID NO: 56 is the amino acid sequence of the V_(H) of the FIB_H1 NHPantibody (VRC 5071).EVQLVQSGAEVKKPGASVKVSCKASGHIFTSYVINWLQEAPGQGFEWMGGIHPGNGGRDYAQKFQGRVTITADMSTSTVYMELRSLRSEDMAVYYCAASSGSYGVSSLDVWGRGVLVTVSS SEQ ID NO: 57 is anexemplary nucleotide sequence encoding the V_(L) of the FIB_H1 NHPantibody (VRC 5072).cagtctgccctgactcagccaccctccctgtctgcatccccgggagcatcggccagactcccctgcaccctgagcagtgacctcagtgttggtagtaaaaacatgtactggtaccagcagaagccagggagcgctcccaggttattcctgtactactactccgactcagacaagcagctgggacctggggtccccaatcgagtctctggctccaaggagacctcaagtaacacagcgtttttgctcatctctgggctccagcctgaggacgaggccgattattactgtcaggtgtatgacagtagtgctaattgggtattcggcggagggacccggctgacagtacta SEQ ID NO: 58is the amino acid sequence of the V_(L) of the FIB_H1 NHP antibody (VRC5072).QSALTQPPSLSASPGASARLPCTLSSDLSVGSKNMYWYQQKPGSAPRLFLYYYSDSDKQLGPGVPNRVSGSKETSSNTAFLLISGLQPEDEADYYCQVYDSSANWVFGGGTRLTVL SEQ ID NOs: 59-109 are aminoacid sequences of heavy and light chain CDRs. SEQ ID NO: 110 is theamino acid sequence of the V_(L) of the C2 antibody with a G29Smutation.DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNSYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPAFGGGTKLEIK SEQ ID NO: 111 is the aminoacid sequence of the V_(L) of the C2 antibody with a G29A mutation.DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNAYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPAFGGGTKLEIK SEQ ID NOs: 112 and 113 areamino acid sequences of light chain CDRs. SEQ ID NO: 114 is an exemplarynucleotide sequence encoding the V_(H) of the G2 antibody.cattcccaggtgcagctgcagcagtctggaggtgagctggtgaagcctggggcttcagtgaagctgtcctgcaagacttctggcttcaccttcagcagtagctatataagttggttgaagcaaaagcctggacagagtcttgagtggattgcatggatttatgctggaactggtggtactgaatataatcagaagttcacaggcaaggcccaagtgactgtagacacatcctccagcacagcctacatgcaattcagcagcctgacaactgaggactctgccatctattactgtgcaagaggaggtagtagcttcgctatggactactggggtcaaggaacctcagtcaccgtctcctca SEQID NO: 115 is the amino acid sequence of the V_(H) of the G2 antibody.QVQLQQSGGELVKPGASVKLSCKTSGFTFSSSYISWLKQKPGQSLEWIAWIYAGTGGTEYNQKFTGKAQVTVDTSSSTAYMQFSSLTTEDSAIYYCARGGSSFAMDYWGQGTSVTVSS SEQ ID NO: 116 is anexemplary nucleotide sequence encoding the V_(L) of the G2 antibody.caacttgtgctgacccaatctccagcttctttggctgtgtctctagggcagagggccaccatctcctgcagagccagcgaaagtgttgataattatggcattagttttatgaactggttccaacagaaaccaggacagccacccaaactcctcatccatactgcatccaaccaaggatccggggtccctgccaggtttagtggcagtgggtctgggacagacttcagcctcaacatccatcctgtggaggacgatgatactgcaatgtatttctgtcagcaaagtgaggaggttcctctcacgttcggtgctgggaccaagctggaaatcaaa SEQ ID NO: 117 is the aminoacid sequence of the V_(L) of the G2 antibody.QLVLTQSPASLAVSLGQRATISCRASESVDNYGISFMNWFQQKPGQPPKLLIHTASNQGSGVPARFSGSGSGTDFSLNIHPVEDDDTAMYFCQQSEEVPLTFGAGTKLELK SEQ ID NO: 118 is an exemplarynucleotide sequence encoding the V_(H) of the G4 antibody.caggtccagctgcagcagtctgggcctgagctggtgaggcctggggtctcagtgaagatttcctgcaagggttccggctacacattcactgattatgctatacactgggtgaagcagagtcatgcaaagagtctagagtggattggggtttttagtacttactatggtaatacaaactacaaccagaagtttaagggcagggccacaatgactgtagacaaatcctccagcacagcctatatggaacttgccagattgacatctgaggattctgccatctattactgtgcaagaaagtcctactatgttgactacgttgatgctatggactactggggtcaaggaacctcagtcaccgtctcctca SEQ ID NO: 119 is the amino acid sequence of the V_(H) of the G4antibody.QVQLQQSGPELVRPGVSVKISCKGSGYTFTDYAIHWVKQSHAKSLEWIGVFSTYYGNTNYNQKFKGRATMTVDKSSSTAYMELARLTSEDSAIYYCARKSYYVDYVDAMDYWGQGTSVTVSS SEQ ID NO: 120 is anexemplary nucleotide sequence encoding the V_(L) of the G4 antibody.gacattgtgctgacccaatctccagcttctttggctgtgtctctagggcagagggccaccatctcctgcagagccagcgaaagtgttgataattatggcattagttttatgaactggttccaacagaaaccaggacagccacccaaactcctcatctctgctacatccaaccaaggatccggggtccctgccaggtttattggcagtgggtctgggacagacttcagcctcaacatccatcctgtggaggaggatgatactgcaatgtatttctgtcagcaaagtaaggaggttcctcggacgttcggtggaggcaccaagctggaaatcaaac SEQ ID NO: 121 is the aminoacid sequence of the V_(L) of the G4 antibody.DIVLTQSPASLAVSLGQRATISCRASESVDNYGISFMNWFQQKPGQPPKLLISATSNQGSGVPARFIGSGSGTDFSLNIHPVEEDDTAMYFCQQSKEVPRTFGGGTKLEIK SEQ ID NO: 122 is an exemplarynucleotide sequence encoding the V_(H) of the D12 antibody.gaggtgaagctggtggagtctgggggaggcttagtgaagcctggagggtccctgaaactctcctgtgcagcctctggattcactttcagtagctatgccatgtcttgggttcgccagactccggagaagaggctggagtgggtcgcaaccattagtagtggtggtacttacacctactatccagacagtgtgaaggggcgattcaccatctccagagacaatgccgagaacaccctgtacctgcaaatgagcagtctgaggtctgaggacacggccatgtattactgtgtaagagatggtaattctatggactactggggtcaaggaacctcagtcaccgtctcctcagc SEQ ID NO:123 is the amino acid sequence of the V_(H) of the D12 antibody.EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPEKRLEWVATISSGGTYTYYPDSVKGRFTISRDNAENTLYLQMSSLRSEDTAMYYCVRDGNSMDYWGQGTSVTVSS SEQ ID NO: 124 is anexemplary nucleotide sequence encoding the V_(L) of the D12 antibody.gatatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcatttgcagggcaagtcaggacattaacaattatttaaactggtatcaacagaaaccagatggaactgttaaactcctgatctactacacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggatcagattattctctcaccattagcaacctggaacaagaagatattgccacttacttttgccaacaggctaatacgcttcctcccacgttcggtgctgggaccaagctggaactgaga SEQ ID NO: 125 is the amino acid sequenceof the V_(L) of the D12 antibody.DIQMTQTTSSLSASLGDRVTIICRASQDINNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGSDYSLTISNLEQEDIATYFCQQANTLPPTFGAGTKLELR SEQ ID NO: 126 is an exemplarynucleotide sequence encoding the V_(H) of the F11 antibody.cattccgaggtgaagctggaggagtctgggggaggcttagtgaagcctggagggtccctgaaactctcctgtgcagcctctggattcactttcagtaggtatgccatgtcttgggttcgccagactccggagaagaggctggagtgggtcgcaaccattaataatggtggtagttacagttactatccagacagtgtgaagggtcgactcaccatctccagagacaatgccaagaacaccctgtacctgcaaatgagcagtctgaggtctgaggacacggccttgtattactgtgcaagacactatgattacgacggatattactatactatggacttctggggtcaaggaacctcagtcaccgtctcctcagc SEQ ID NO: 127 is the amino acid sequence of the V_(H) of theF11 antibody.EVKLEESGGGLVKPGGSLKLSCAASGFTFSRYAMSWVRQTPEKRLEWVATINNGGSYSYYPDSVKGRLTISRDNAKNTLYLQMSSLRSEDTALYYCARHYDYDGYYYTMDFWGQGTSVTVSS SEQ ID NO: 128 is anexemplary nucleotide sequence encoding the V_(L) of the F11 antibody.gatgttttgatgacccaaattccactctccctgcctgtcagtcttggagatcaagcctccatttcttgcagatctagtcagagcattgtacatagtaatggaaacacctatttagaatggtacctgcagaaaccaggccagtctccaaagcccctgatctacaaagtttccaaccgaatttctggggtcccagacaggttcagtggcagtggatcagggacagatttcacactcaagatcagcagagtggaggctgaggatctgggagtttattactgctttcaaggttcacatgttccgtacacgttcggaggggggaccaacctggaaataaaacg SEQ ID NO: 129 is theamino acid sequence of the V_(L) of the F11 antibody.DVLMTQIPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKPLIYKVSNRISGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTNLEIKR SEQ ID NOs: 130-152 are aminoacid sequences of heavy and light chain CDRs. SEQ ID NO: 153 is anexemplary nucleotide sequence encoding a chimeric V_(H) including the G2heavy chain variable domain and a human IgG1 constant domain (VRC 5068).atgggatggtcatgtatcatcctttttctagtagcaactgcaaccggtgtacattcccaggtgcagctgcagcagtctggaggtgagctggtgaagcctggggcttcagtgaagctgtcctgcaagacttctggcttcaccttcagcagtagctatataagttggttgaagcaaaagcctggacagagtcttgagtggattgcatggatttatgctggaactggtggtactgaatataatcagaagttcacaggcaaggcccaagtgactgtagacacatcctccagcacagcctacatgcaattcagcagcctgacaactgaggactctgccatctattactgtgcaagaggaggtagtagcttcgctatggactactggggtcaaggaacctcagtcaccgtctcctcagcgtcgaccacgcccccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaacccgtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgaSEQ ID NO: 154 is a chimeric V_(H) including the G2 heavy chain variabledomain and a human IgG1 constant domain (VRC 5068).MGWSCIILFLVATATGVHSQVQLQQSGGELVKPGASVKLSCKTSGFTFSSSYISWLKQKPGQSLEWIAWIYAGTGGTEYNQKFTGKAQVTVDTSSSTAYMQFSSLTTEDSAIYYCARGGSSFAMDYWGQGTSVTVSSASTTPPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

DETAILED DESCRIPTION

MERS-CoV has emerged as a highly fatal cause of severe acute respiratoryinfection. The high case fatality rate, vaguely defined epidemiology,and absence of prophylactic or therapeutic measures against this novelvirus have created an urgent need for an effective vaccine, should theoutbreak expand to pandemic proportions.

Past efforts to develop coronavirus vaccines have used whole-inactivatedvirus, live-attenuated virus, recombinant protein subunit, or geneticapproaches (Graham et al., Nature reviews. Microbiology 11, 836, 2013).This disclosure provides an immunization strategy based on the MERS-CoVSpike glycoprotein (S). In one non-limiting embodiment, a prime-boostimmunization strategy including a full-length S DNA prime and S1 subunitprotein boost was identified to elicit high titers of neutralizingantibodies against several different MERS-CoV strains. Immunization withDNA expressing full-length S followed by S1 subunit protein yieldedpotent neutralizing mAbs in both mice and NHPs. Compared to proteinalone, S DNA prime/S1 protein boost immunization yielded a morefunctionally diverse repertoire of neutralizing antibodies and alsogenerated a Th1-biased immune response.

Vaccine-elicited murine monoclonal antibodies were also identified andshown to neutralize virus by targeting the receptor binding domain(RBD), multiple non-RBD portions of S1, or S2.

I. Summary of Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes X, published by Jones & BartlettPublishers, 2009; and Meyers et al. (eds.), The Encyclopedia of CellBiology and Molecular Medicine, published by Wiley-VCH in 16 volumes,2008; and other similar references.

As used herein, the singular forms “a,” “an,” and “the,” refer to boththe singular as well as plural, unless the context clearly indicatesotherwise. For example, the term “an antigen” includes single or pluralantigens and can be considered equivalent to the phrase “at least oneantigen.” As used herein, the term “comprises” means “includes.” It isfurther to be understood that any and all base sizes or amino acidsizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescriptive purposes, unless otherwise indicated. Although many methodsand materials similar or equivalent to those described herein can beused, particular suitable methods and materials are described herein. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting. To facilitatereview of the various embodiments, the following explanations of termsare provided:

Adjuvant: A vehicle used to enhance antigenicity. Adjuvants include asuspension of minerals (alum, aluminum hydroxide, or phosphate) on whichantigen is adsorbed; or water-in-oil emulsion, for example, in whichantigen solution is emulsified in mineral oil (Freund incompleteadjuvant), sometimes with the inclusion of killed mycobacteria (Freund'scomplete adjuvant) to further enhance antigenicity (inhibits degradationof antigen and/or causes influx of macrophages). Immunostimulatoryoligonucleotides (such as those including a CpG motif) can also be usedas adjuvants. Adjuvants include biological molecules (a “biologicaladjuvant”), such as costimulatory molecules. Exemplary adjuvants includeIL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2,OX-40L, 4-1BBL and toll-like receptor (TLR) agonists, such as TLR-9agonists. The person of ordinary skill in the art is familiar withadjuvants (see, e.g., Singh (ed.) Vaccine Adjuvants and DeliverySystems. Wiley-Interscience, 2007). Adjuvants can be used in combinationwith the disclosed MERS-CoV immunogens.

Administration: The introduction of a composition into a subject by achosen route. Administration can be local or systemic. For example, ifthe chosen route is intravenous, the composition (such as a compositionincluding a disclosed immunogen or antibody) is administered byintroducing the composition into a vein of the subject. Exemplary routesof administration include, but are not limited to, oral, injection (suchas subcutaneous, intramuscular, intradermal, intraperitoneal, andintravenous), sublingual, rectal, transdermal (for example, topical),intranasal, vaginal, and inhalation routes.

Agent: Any substance or any combination of substances that is useful forachieving an end or result; for example, a substance or combination ofsubstances useful for inhibiting MERS-CoV infection in a subject. Agentsinclude proteins, nucleic acid molecules, compounds, small molecules,organic compounds, inorganic compounds, or other molecules of interest.An agent can include a therapeutic agent (such as an anti-retroviralagent), a diagnostic agent or a pharmaceutical agent. In someembodiments, the agent is a protein agent (such as a recombinantMERS-CoV polypeptide or immunogenic fragment thereof, orMERS-CoV-specific antibody), or an anti-viral agent. The skilled artisanwill understand that particular agents may be useful to achieve morethan one result.

Amino acid substitution: The replacement of one amino acid in apolypeptide with a different amino acid or with no amino acid (i.e., adeletion). In some examples, an amino acid in a polypeptide issubstituted with an amino acid from a homologous polypeptide, forexample, and amino acid in a recombinant MERS-CoV polypeptide can besubstituted with the corresponding amino acid from a different MERS-CoVstrain.

Antibody: A polypeptide that specifically binds and recognizes ananalyte (antigen) such as MERS-CoV S protein or an antigenic fragmentthereof. The term “antibody” is used herein in the broadest sense andencompasses various antibody structures, including but not limited tomonoclonal antibodies, polyclonal antibodies, multispecific antibodies(e.g., bispecific antibodies), and antigen binding fragments thereof, solong as they exhibit the desired antigen-binding activity. Non-limitingexamples of antibodies include, for example, intact immunoglobulins andvariants and fragments thereof known in the art that retain bindingaffinity for the antigen.

A “monoclonal antibody” is an antibody obtained from a population ofsubstantially homogeneous antibodies, i.e., the individual antibodiescomprising the population are identical except for possible naturallyoccurring mutations that may be present in minor amounts. Monoclonalantibodies are highly specific, being directed against a singleantigenic epitope. The modifier “monoclonal” indicates the character ofthe antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. In some examples, amonoclonal antibody is an antibody produced by a single clone ofB-lymphocytes or by a cell into which nucleic acid encoding the lightand heavy variable regions of the antibody of a single antibody (or anantigen binding fragment thereof) have been transfected, or a progenythereof. In some examples monoclonal antibodies are isolated from asubject. Monoclonal antibodies can have conservative amino acidsubstitutions which have substantially no effect on antigen binding orother immunoglobulin functions. Exemplary methods of production ofmonoclonal antibodies are known, for example, see Harlow & Lane,Antibodies, A Laboratory Manual, 2^(nd) ed. Cold Spring HarborPublications, New York (2013).)

Typically, an immunoglobulin has heavy (H) chains and light (L) chainsinterconnected by disulfide bonds. Immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon and mu constant regiongenes, as well as the myriad immunoglobulin variable domain genes. Thereare two types of light chain, lambda (λ) and kappa (κ). There are fivemain heavy chain classes (or isotypes) which determine the functionalactivity of an antibody molecule: IgM, IgD, IgG, IgA and IgE.

Each heavy and light chain contains a constant region (or constantdomain) and a variable region (or variable domain; see, e.g., Kindt etal. Kuby Immunology, 6.sup.th ed., W.H. Freeman and Co., page 91(2007).) In several embodiments, the heavy and the light chain variableregions combine to specifically bind the antigen. In additionalembodiments, only the heavy chain variable region is required. Forexample, naturally occurring camelid antibodies consisting of a heavychain only are functional and stable in the absence of light chain (see,e.g., Hamers-Casterman et al., Nature, 363:446-448, 1993; Sheriff etal., Nat. Struct. Biol., 3:733-736, 1996). References to “V_(H)” or “VH”refer to the variable region of an antibody heavy chain, including thatof an antigen binding fragment, such as Fv, scFv, dsFv or Fab.References to “V_(L)” or “VL” refer to the variable domain of anantibody light chain, including that of an Fv, scFv, dsFv or Fab.

Light and heavy chain variable regions contain a “framework” regioninterrupted by three hypervariable regions, also called“complementarity-determining regions” or “CDRs” (see, e.g., Kabat etal., Sequences of Proteins of Immunological Interest, U.S. Department ofHealth and Human Services, 1991). The sequences of the framework regionsof different light or heavy chains are relatively conserved within aspecies. The framework region of an antibody, that is the combinedframework regions of the constituent light and heavy chains, serves toposition and align the CDRs in three-dimensional space.

The CDRs are primarily responsible for binding to an epitope of anantigen. The amino acid sequence boundaries of a given CDR can bereadily determined using any of a number of well-known schemes,including those described by Kabat et al. (“Sequences of Proteins ofImmunological Interest,” 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md., 1991; “Kabat” numbering scheme),Al-Lazikani et al., (JMB 273,927-948, 1997; “Chothia” numbering scheme),and Lefranc et al. (“IMGT unique numbering for immunoglobulin and T cellreceptor variable domains and Ig superfamily V-like domains,” Dev. Comp.Immunol., 27:55-77, 2003; “IMGT” numbering scheme). The CDRs of eachchain are typically referred to as CDR1, CDR2, and CDR3 (from theN-terminus to C-terminus), and are also typically identified by thechain in which the particular CDR is located. Thus, a V_(H) CDR3 is theCDR3 from the variable domain of the heavy chain of the antibody inwhich it is found, whereas a V_(L) CDR1 is the CDR1 from the variabledomain of the light chain of the antibody in which it is found. Lightchain CDRs are sometimes referred to as LCDR1, LCDR2, and LCDR3. Heavychain CDRs are sometimes referred to as HCDR1, HCDR2, and HCDR3.

An “antigen binding fragment” is a portion of a full length antibodythat retains the ability to specifically recognize the cognate antigen,as well as various combinations of such portions. Non-limiting examplesof antigen binding fragments include Fv, Fab, Fab′, Fab′-SH, F(ab)₂;diabodies; linear antibodies; single-chain antibody molecules (e.g.scFv); and multispecific antibodies formed from antibody fragments.Antibody fragments include antigen binding fragments either produced bythe modification of whole antibodies or those synthesized de novo usingrecombinant DNA methodologies (see, e.g., Kontermann and Dubel (Ed),Antibody Engineering, Vols. 1-2, 2^(nd) Ed., Springer Press, 2010).

A single-chain antibody (scFv) is a genetically engineered moleculecontaining the V_(H) and V_(L) domains of one or more antibody(ies)linked by a suitable polypeptide linker as a genetically fused singlechain molecule (see, for example, Bird et al., Science, 242:423-426,1988; Huston et al., Proc. Natl. Acad. Sci., 85:5879-5883, 1988; Ahmadet al., Clin. Dev. Immunol., 2012, doi:10.1155/2012/980250; Marbry,IDrugs, 13:543-549, 2010). The intramolecular orientation of theV_(H)-domain and the V_(L)-domain in a scFv, is typically not decisivefor scFvs. Thus, scFvs with both possible arrangements(V_(H)-domain-linker domain-V_(L)-domain; V_(L)-domain-linkerdomain-V_(H)-domain) may be used.

In a dsFv the heavy and light chain variable chains have been mutated tointroduce a disulfide bond to stabilize the association of the chains.Diabodies also are included, which are bivalent, bispecific antibodiesin which V_(H) and V_(L) domains are expressed on a single polypeptidechain, but using a linker that is too short to allow for pairing betweenthe two domains on the same chain, thereby forcing the domains to pairwith complementary domains of another chain and creating two antigenbinding sites (see, for example, Holliger et al., Proc. Natl. Acad.Sci., 90:6444-6448, 1993; Poljak et al., Structure, 2:1121-1123, 1994).

Antibodies also include genetically engineered forms such as chimericantibodies (such as humanized murine antibodies) and heteroconjugateantibodies (such as bispecific antibodies). See also, Pierce Catalog andHandbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J.,Immunology, 3^(rd) Ed., W.H. Freeman & Co., New York, 1997.

Non-naturally occurring antibodies can be constructed using solid phasepeptide synthesis, can be produced recombinantly, or can be obtained,for example, by screening combinatorial libraries consisting of variableheavy chains and variable light chains as described by Huse et al.,Science 246:1275-1281 (1989), which is incorporated herein by reference.These and other methods of making, for example, chimeric, humanized,CDR-grafted, single chain, and bifunctional antibodies, are well knownto those skilled in the art (Winter and Harris, Immunol. Today14:243-246 (1993); Ward et al., Nature 341:544-546 (1989); Harlow andLane, supra, 1988; Hilyard et al., Protein Engineering: A practicalapproach (IRL Press 1992); Borrabeck, Antibody Engineering, 2d ed.(Oxford University Press 1995); each of which is incorporated herein byreference).

An “antibody that binds to the same epitope” as a reference antibodyrefers to an antibody that blocks binding of the reference antibody toits antigen in a competition assay by 50% or more, and conversely, thereference antibody blocks binding of the antibody to its antigen in acompetition assay by 50% or more. Antibody competition assays are known,and an exemplary competition assay is provided herein.

A “humanized” antibody or antigen binding fragment includes a humanframework region and one or more CDRs from a non-human (such as a mouse,rat, or synthetic) antibody or antigen binding fragment. The non-humanantibody or antigen binding fragment providing the CDRs is termed a“donor,” and the human antibody or antigen binding fragment providingthe framework is termed an “acceptor.” In one embodiment, all the CDRsare from the donor immunoglobulin in a humanized immunoglobulin.Constant regions need not be present, but if they are, they can besubstantially identical to human immunoglobulin constant regions, suchas at least about 85-90%, such as about 95% or more identical. Hence,all parts of a humanized antibody or antigen binding fragment, exceptpossibly the CDRs, are substantially identical to corresponding parts ofnatural human antibody sequences.

A “chimeric antibody” is an antibody which includes sequences derivedfrom two different antibodies, which typically are of different species.In some examples, a chimeric antibody includes one or more CDRs and/orframework regions from one human antibody and CDRs and/or frameworkregions from another human antibody.

A “fully human antibody” or “human antibody” is an antibody whichincludes sequences from (or derived from) the human genome, and does notinclude sequence from another species. In some embodiments, a humanantibody includes CDRs, framework regions, and (if present) an Fc regionfrom (or derived from) the human genome. Human antibodies can beidentified and isolated using technologies for creating antibodies basedon sequences derived from the human genome, for example by phage displayor using transgenic animals (see, e.g., Barbas et al. Phage display: ALaboratory Manuel. 1″ Ed. New York: Cold Spring Harbor Laboratory Press,2004. Print.; Lonberg, Nat. Biotech., 23: 1117-1125, 2005; Lonenberg,Curr. Opin. Immunol., 20:450-459, 2008).

An antibody may have one or more binding sites. If there is more thanone binding site, the binding sites may be identical to one another ormay be different. For instance, a naturally-occurring immunoglobulin hastwo identical binding sites, a single-chain antibody or Fab fragment hasone binding site, while a bispecific or bifunctional antibody has twodifferent binding sites.

Antigen: A compound, composition, or substance that can stimulate theproduction of antibodies or a T cell response in an animal, includingcompositions that are injected or absorbed into an animal. An antigenreacts with the products of specific humoral or cellular immunity,including those induced by heterologous antigens, such as the disclosedMERS-CoV antigens. Examples of antigens include, but are not limited to,polypeptides, peptides, lipids, polysaccharides, combinations thereof(such as glycopeptides) and nucleic acids containing antigenicdeterminants, such as those recognized by an immune cell. In someexamples, antigens include peptides derived from a pathogen of interest,such as MERS-CoV. An antigen can include one or more epitopes.

Codon-optimized: A “codon-optimized” nucleic acid refers to a nucleicacid sequence that has been altered such that the codons are optimal forexpression in a particular system (such as a particular species or groupof species). For example, a nucleic acid sequence can be optimized forexpression in mammalian (such as human) cells. Codon optimization doesnot alter the amino acid sequence of the encoded protein.

Conditions sufficient to form an immune complex: Conditions which allowan antibody or antigen binding fragment thereof to bind to its cognateepitope to a detectably greater degree than, and/or to the substantialexclusion of, binding to substantially all other epitopes. Conditionssufficient to form an immune complex are dependent upon the format ofthe binding reaction and typically are those utilized in immunoassayprotocols or those conditions encountered in vivo. See Harlow & Lane,Antibodies, A Laboratory Manual, 2^(nd) ed. Cold Spring HarborPublications, New York (2013) for a description of immunoassay formatsand conditions. The conditions employed in the methods are“physiological conditions” which include reference to conditions (e.g.,temperature, osmolarity, pH) that are typical inside a living mammal ora mammalian cell. While it is recognized that some organs are subject toextreme conditions, the intra-organismal and intracellular environmentnormally lies around pH 7 (e.g., from pH 6.0 to pH 8.0, more typicallypH 6.5 to 7.5), contains water as the predominant solvent, and exists ata temperature above 0° C. and below 50° C. Osmolarity is within therange that is supportive of cell viability and proliferation.

Conjugate: A complex of two molecules linked together, for example,linked together by a covalent bond. In some embodiment, an antibody islinked to an effector molecule or detectable marker; for example, anantibody that specifically binds to MERS-CoV covalently linked to aneffector molecule or detectable marker. The linkage can be by chemicalor recombinant means. In one embodiment, the linkage is chemical,wherein a reaction between the antibody moiety and the effector moleculehas produced a covalent bond formed between the two molecules to formone molecule. A peptide linker (short peptide sequence) can optionallybe included between the antibody and the effector molecule. Becauseconjugates can be prepared from two molecules with separatefunctionalities, such as an antibody and an effector molecule, they arealso sometimes referred to as “chimeric molecules.”

Conservative variants: “Conservative” amino acid substitutions are thosesubstitutions that do not substantially affect or decrease a function ofa protein, such as the ability of the protein to induce an immuneresponse when administered to a subject. For example, in someembodiments, a recombinant MERS-CoV S protein or S1 fragment can includeup to 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 conservative substitutionscompared to a corresponding native MERS-CoV protein sequence and inducean immune response to MERS-CoV S protein in a subject. The termconservative variation also includes the use of a substituted amino acidin place of an unsubstituted parent amino acid.

Furthermore, one of ordinary skill will recognize that individualsubstitutions, deletions or additions which alter, add or delete asingle amino acid or a small percentage of amino acids (for instanceless than 5%, in some embodiments less than 1%) in an encoded sequenceare conservative variations where the alterations result in thesubstitution of an amino acid with a chemically similar amino acid.

Conservative amino acid substitution tables providing functionallysimilar amino acids are well known to one of ordinary skill in the art.The following six groups are examples of amino acids that are consideredto be conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Non-conservative substitutions are those that reduce an activity orfunction of protein, e.g., a MERS-CoV S protein, such as the ability toinduce an immune response when administered to a subject. For instance,if an amino acid residue is essential for a function of the protein,even an otherwise conservative substitution may disrupt that activity.Thus, a conservative substitution does not alter the basic function of aprotein of interest.

Contacting: Placement in direct physical association; includes both insolid and liquid form, which can take place either in vivo or in vitro.Contacting includes contact between one molecule and another molecule,for example the amino acid on the surface of one polypeptide, such as apeptide, that contacts another polypeptide. Contacting can also includecontacting a cell for example by placing a polypeptide in directphysical association with a cell.

Control: A reference standard. In some embodiments, the control is anegative control sample obtained from a healthy patient. In otherembodiments, the control is a positive control sample obtained from apatient diagnosed with MERS-CoV infection. In still other embodiments,the control is a historical control or standard reference value or rangeof values (such as a previously tested control sample, such as a groupof MERS-CoV patients with known prognosis or outcome, or group ofsamples that represent baseline or normal values).

A difference between a test sample and a control can be an increase orconversely a decrease. The difference can be a qualitative difference ora quantitative difference, for example a statistically significantdifference. In some examples, a difference is an increase or decrease,relative to a control, of at least about 5%, such as at least about 10%,at least about 20%, at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, at least about 100%, at least about 150%, at leastabout 200%, at least about 250%, at least about 300%, at least about350%, at least about 400%, at least about 500%, or greater than 500%.

Degenerate variant: In the context of the present disclosure, a“degenerate variant” refers to a polynucleotide encoding a polypeptide(such as a MERS-CoV S protein or immunogenic fragment thereof) thatincludes a sequence that is degenerate as a result of the genetic code.There are 20 natural amino acids, most of which are specified by morethan one codon. Therefore, all degenerate nucleotide sequences encodinga peptide are included as long as the amino acid sequence of the peptideencoded by the nucleotide sequence is unchanged.

Detectable marker: A detectable molecule (also known as a label) that isconjugated directly or indirectly to a second molecule, such as anantibody, to facilitate detection of the second molecule. For example,the detectable marker can be capable of detection by ELISA,spectrophotometry, flow cytometry, microscopy or diagnostic imagingtechniques (such as CT scans, MRIs, ultrasound, fiberoptic examination,and laparoscopic examination). Specific, non-limiting examples ofdetectable markers include fluorophores, chemiluminescent agents,enzymatic linkages, radioactive isotopes and heavy metals or compounds(for example super paramagnetic iron oxide nanocrystals for detection byMRI). In one example, a “labeled antibody” refers to incorporation ofanother molecule in the antibody. For example, the label is a detectablemarker, such as the incorporation of a radiolabeled amino acid orattachment to a polypeptide of biotinyl moieties that can be detected bymarked avidin (for example, streptavidin containing a fluorescent markeror enzymatic activity that can be detected by optical or colorimetricmethods). Various methods of labeling polypeptides and glycoproteins areknown in the art and may be used. Examples of labels for polypeptidesinclude, but are not limited to, the following: radioisotopes orradionuclides (such as ³⁵S or ¹³¹I), fluorescent labels (such asfluorescein isothiocyanate (FITC), rhodamine, lanthanide phosphors),enzymatic labels (such as horseradish peroxidase, beta-galactosidase,luciferase, alkaline phosphatase), chemiluminescent markers, biotinylgroups, predetermined polypeptide epitopes recognized by a secondaryreporter (such as a leucine zipper pair sequences, binding sites forsecondary antibodies, metal binding domains, epitope tags), or magneticagents, such as gadolinium chelates. In some embodiments, labels areattached by spacer arms of various lengths to reduce potential sterichindrance. Methods for using detectable markers and guidance in thechoice of detectable markers appropriate for various purposes arediscussed for example in Sambrook et al. (Molecular Cloning: ALaboratory Manual, 4^(th) ed, Cold Spring Harbor, N.Y., 2012) andAusubel et al. (In Current Protocols in Molecular Biology, John Wiley &Sons, New York, through supplement 104, 2013).

Detecting: To identify the existence, presence, or fact of something.General methods of detecting are known to the skilled artisan and may besupplemented with the protocols and reagents disclosed herein. Forexample, included herein are methods of detecting the level of a proteinin a sample or a subject.

Effector molecule: The portion of a chimeric molecule that is intendedto have a desired effect on a cell to which the chimeric molecule istargeted. Effector molecule is also known as an effector moiety (EM),therapeutic agent, or diagnostic agent, or similar terms.

Dipeptidyl peptidase 4 (DPP4): A 240 kDa homodimeric, type II membraneglycoprotein that serves as the cellular receptor for MERS-CoV. DPP4 isdistributed in most mammalian tissues, and highly expressed in kidney,liver and endothelium. DPP4 includes a short cytoplasmic domain, atransmembrane domain and a relatively large extracellular (ectodomain)sequence of about 740 amino acids. The RBD of MERS-CoV S protein bindsto the extracellular domain of DPP4 before fusion with target cellmembrane. An exemplary human DPP4 amino acid sequence is provided inGenBank as deposit No. NP_001926.2, which is incorporated by referenceherein in its entirety.

Epitope: An antigenic determinant. These are particular chemical groupsor peptide sequences on a molecule that are antigenic, such that theyelicit a specific immune response, for example, an epitope is the regionof an antigen to which B and/or T cells respond. An antibody can bind toa particular antigenic epitope, such as an epitope on MERS-CoV Sprotein.

Expression: Transcription or translation of a nucleic acid sequence. Forexample, a gene is expressed when its DNA is transcribed into an RNA orRNA fragment, which in some examples is processed to become mRNA. A genemay also be expressed when its mRNA is translated into an amino acidsequence, such as a protein or a protein fragment. In a particularexample, a heterologous gene is expressed when it is transcribed into anRNA. In another example, a heterologous gene is expressed when its RNAis translated into an amino acid sequence. The term “expression” is usedherein to denote either transcription or translation. Regulation ofexpression can include controls on transcription, translation, RNAtransport and processing, degradation of intermediary molecules such asmRNA, or through activation, inactivation, compartmentalization ordegradation of specific protein molecules after they are produced.

Expression Control Sequences: Nucleic acid sequences that regulate theexpression of a heterologous nucleic acid sequence to which it isoperatively linked. Expression control sequences are operatively linkedto a nucleic acid sequence when the expression control sequences controland regulate the transcription and, as appropriate, translation of thenucleic acid sequence. Thus expression control sequences can includeappropriate promoters, enhancers, transcription terminators, a startcodon (ATG) in front of a protein-encoding gene, splicing signal forintrons, maintenance of the correct reading frame of that gene to permitproper translation of mRNA, and stop codons. The term “controlsequences” is intended to include, at a minimum, components whosepresence can influence expression, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences. Expression control sequences can include apromoter.

A promoter is a minimal sequence sufficient to direct transcription.Also included are those promoter elements which are sufficient to renderpromoter-dependent gene expression controllable for cell-type specific,tissue-specific, or inducible by external signals or agents; suchelements may be located in the 5′ or 3′ regions of the gene. Bothconstitutive and inducible promoters are included (see for example,Bitter et al., Methods in Enzymology 153:516-544, 1987). For example,when cloning in bacterial systems, inducible promoters such as pL ofbacteriophage lambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) andthe like may be used. In one embodiment, when cloning in mammalian cellsystems, promoters derived from the genome of mammalian cells (such asmetallothionein promoter) or from mammalian viruses (such as theretrovirus long terminal repeat; the adenovirus late promoter; thevaccinia virus 7.5K promoter) can be used. Promoters produced byrecombinant DNA or synthetic techniques may also be used to provide fortranscription of the nucleic acid sequences.

A polynucleotide can be inserted into an expression vector that containsa promoter sequence which facilitates the efficient transcription of theinserted genetic sequence of the host. The expression vector typicallycontains an origin of replication, a promoter, as well as specificnucleic acid sequences that allow phenotypic selection of thetransformed cells.

Expression vector: A vector comprising a recombinant polynucleotidecomprising expression control sequences operatively linked to anucleotide sequence to be expressed. An expression vector comprisessufficient cis-acting elements for expression; other elements forexpression can be supplied by the host cell or in an in vitro expressionsystem. Expression vectors include all those known in the art, such ascosmids, plasmids (e.g., naked or contained in liposomes) and viruses(e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associatedviruses) that incorporate the recombinant polynucleotide.

Heterologous: Originating from a different genetic source. A nucleicacid molecule that is heterologous to a cell originated from a geneticsource other than the cell in which it is expressed. In one specific,non-limiting example, a heterologous nucleic acid molecule encoding arecombinant MERS-CoV polypeptide or specific antibody is expressed in acell, such as a mammalian cell. Methods for introducing a heterologousnucleic acid molecule in a cell or organism are well known in the art,for example transformation with a nucleic acid, includingelectroporation, lipofection, particle gun acceleration, and homologousrecombination.

Ferritin: A protein that stores iron and releases it in a controlledfashion. The protein is produced by almost all living organisms.Ferritin polypeptides assemble into a globular protein complex of 24protein subunits, each of the 24 subunits includes a single ferritinpolypeptide. In some examples, ferritin is used to form a nanoparticlepresenting antigens on its surface, for example, an MERS-CoV antigen.

Host cells: Cells in which a vector can be propagated and its DNAexpressed. The cell may be prokaryotic or eukaryotic. The term alsoincludes any progeny of the subject host cell. It is understood that allprogeny may not be identical to the parental cell since there may bemutations that occur during replication. However, such progeny areincluded when the term “host cell” is used.

IgA: A polypeptide belonging to the class of antibodies that aresubstantially encoded by a recognized immunoglobulin alpha gene. Inhumans, this class or isotype comprises IgA₁ and IgA₂. IgA antibodiescan exist as monomers, polymers (referred to as pIgA) of predominantlydimeric form, and secretory IgA. The constant chain of wild-type IgAcontains an 18-amino-acid extension at its C-terminus called the tailpiece (tp). Polymeric IgA is secreted by plasma cells with a 15-kDapeptide called the J chain linking two monomers of IgA through theconserved cysteine residue in the tail piece.

IgG: A polypeptide belonging to the class or isotype of antibodies thatare substantially encoded by a recognized immunoglobulin gamma gene. Inhumans, this class comprises IgG₁, IgG₂, IgG₃, and IgG₄. In mice, thisclass comprises IgG₁, IgG_(2a), IgG_(2b), IgG₃.

Immune complex: The binding of antibody or antigen binding fragment(such as a scFv) to a soluble antigen forms an immune complex. Theformation of an immune complex can be detected through conventionalmethods known to the skilled artisan, for instance immunohistochemistry,immunoprecipitation, flow cytometry, immunofluorescence microscopy,ELISA, immunoblotting (for example, Western blot), magnetic resonanceimaging, CT scans, X-ray and affinity chromatography. Immunologicalbinding properties of selected antibodies may be quantified usingmethods well known in the art.

Immune response: A response of a cell of the immune system, such as a Bcell, T cell, or monocyte, to a stimulus. In one embodiment, theresponse is specific for a particular antigen (an “antigen-specificresponse”). In one embodiment, an immune response is a T cell response,such as a CD4+ response or a CD8+ response. In another embodiment, theresponse is a B cell response, and results in the production of specificantibodies. “Enhancing an immune response” refers to co-administrationof an adjuvant and an immunogenic agent, wherein the adjuvant increasesthe desired immune response to the immunogenic agent compared toadministration of the immunogenic agent to the subject in the absence ofthe adjuvant.

Immunogen: A protein or a portion thereof that is capable of inducing animmune response in a mammal, such as a mammal infected or at risk ofinfection with a pathogen. Administration of an immunogen or nucleicacid encoding the immunogen can lead to protective immunity and/orproactive immunity against a pathogen of interest. In some examples, animmunogen is a recombinant MERS-CoV S protein as disclosed herein.

Immunogenic composition: A composition comprising an immunogenicpolypeptide, or a nucleic acid molecule or vector encoding animmunogenic polypeptide that induces a measurable CTL response againstthe immunogenic polypeptide, or induces a measurable B cell response(such as production of antibodies) against the immunogenic polypeptide.In one example, an “immunogenic composition” is a composition thatincludes a disclosed MERS-CoV S protein or immunogenic fragment thereof,that induces a measurable CTL response against MERS-CoV S protein, orinduces a measurable B cell response (such as production of antibodies)against a MERS-CoV S protein. It further refers to isolated nucleicacids encoding an antigen, such as a nucleic acid that can be used toexpress the antigen (and thus be used to elicit an immune responseagainst this peptide).

For in vitro use, an immunogenic composition may comprise or consist ofthe isolated protein or nucleic acid molecule encoding the protein. Forin vivo use, the immunogenic composition will typically include theprotein or nucleic acid molecule in a pharmaceutically acceptablecarrier and may also include other agents, such as an adjuvant. Anyparticular protein, such as a MERS-CoV S protein or a nucleic acidencoding the protein, can be readily tested for its ability to induce aCTL or B cell response by art-recognized assays. Immunogeniccompositions can include adjuvants, which are well known to one of skillin the art.

Isolated: An “isolated” biological component (such as a protein, forexample a disclosed immunogen or nucleic acid encoding such an antigen)has been substantially separated or purified away from other biologicalcomponents, such as other biological components in which the componentnaturally occurs, such as other chromosomal and extrachromosomal DNA,RNA, and proteins. Proteins, peptides and nucleic acids that have been“isolated” include proteins purified by standard purification methods.The term also embraces proteins or peptides prepared by recombinantexpression in a host cell as well as chemically synthesized proteins,peptides and nucleic acid molecules. Isolated does not require absolutepurity, and can include protein, peptide, or nucleic acid molecules thatare at least 50% isolated, such as at least 75%, 80%, 90%, 95%, 98%,99%, or even 99.9% isolated.

K_(D): The dissociation constant for a given interaction, such as apolypeptide ligand interaction or an antibody antigen interaction. Forexample, for the bimolecular interaction of an antibody or antigenbinding fragment and an immunogen (such as MERS-CoV S protein) it is theconcentration of the individual components of the bimolecularinteraction divided by the concentration of the complex.

Linker: A bi-functional molecule that can be used to link two moleculesinto one contiguous molecule, for example, to link a carrier molecule toa immunogenic polypeptide. Non-limiting examples of peptide linkersinclude glycine-serine linkers, such as a (GGGGS, SEQ ID NO: 155)_(x)linker.

The terms “conjugating,” “joining,” “bonding,” or “linking” can refer tomaking two molecules into one contiguous molecule; for example, linkingtwo polypeptides into one contiguous polypeptide, or covalentlyattaching a carrier molecule or other molecule to an immunogenicpolypeptide, such as an MERS-CoV S protein as disclosed herein. Thelinkage can be either by chemical or recombinant means. “Chemical means”refers to a reaction, for example, between the immunogenic polypeptidemoiety and the carrier molecule such that there is a covalent bondformed between the two molecules to form one molecule.

Middle East respiratory syndrome coronavirus (MERS-CoV): Apositive-sense, single stranded RNA virus of the genus Betacoronavirusthat has emerged as a highly fatal cause of severe acute respiratoryinfection. The viral genome is capped, polyadenylated, and covered withnucleocapsid proteins. The MERS-CoV virion includes a viral envelopewith large spike glycoproteins. The MERS-CoV genome, like mostcoronaviruses, has a common genome organization with the replicase geneincluded in the 5′-two thirds of the genome, and structural genesincluded in the 3′-third of the genome. The MERS-CoV genome encodes thecanonical set of structural protein genes in the order 5′-spike(S)-envelope (E)-membrane (M) and nucleocapsid (N)-3′. MERS-CoV genomesare currently classified as Clade A or Clade B MERS-CoV. Examples ofClade A and Clade B viruses include the JordanN3/2012 (GenBank ID:KC776174) and England_Qatar/2012 (GenBank ID: KC667074) strains,respectively.

Methods of identifying a subject with a MERS-CoV infection are known inthe art and include (see, e.g., Sampathkumar P, Mayo Clin Proc. 2014August; 89(8):1153-8).

MERS-CoV Spike (5) protein: A class I fusion glycoprotein, the MERS-CoVS protein is initially synthesized as a precursor protein ofapproximately 1350 amino acids in size. Individual precursor Spolypeptides form a homotrimer and undergo glycosylation within theGolgi apparatus as well as processing to remove the signal peptide, andcleavage by a cellular protease between approximately position 752/753to generate separate S1 and S2 polypeptide chains, which remainassociated as S1/S2 protomers within the homotrimer. The S1 subunit isdistal to the virus membrane and contains the receptor-binding domain(RBD) that mediates virus attachment to its host receptor, dipeptidylpeptidase-4 (DPP4), and includes approximately residues 367-606 of the Sprotein. The S2 subunit contains a transmembrane domain and twoheptad-repeat sequences typical of fusion glycoproteins.

The numbering used in the disclosed MERS-CoV S proteins and fragmentsthereof is relative to the S protein of the England1 strain of MERS-CoV,the sequence of which is provided as SEQ ID NO: 14, and deposited inGenBank as No. AFY13307.1, which is incorporated by reference herein inits entirety.

Neutralizing antibody: An antibody which reduces the infectious titer ofan infectious agent by binding to a specific antigen on the infectiousagent. In some examples the infectious agent is a virus. In someexamples, an antibody that is specific for MERS-CoV S proteinneutralizes the infectious titer of MERS-CoV. A “broadly neutralizingantibody” is an antibody that binds to and inhibits the function ofrelated antigens, such as antigens that share at least 85%, 90%, 95%,96%, 97%, 98% or 99% identity antigenic surface of antigen. With regardto an antigen from a pathogen, such as a virus, the antibody can bind toand inhibit the function of an antigen from more than one class and/orsubclass of the pathogen. For example, with regard to MERS-CoV, theantibody can bind to and inhibit the function of an antigen, such asMERS-CoV S protein from more than one strain of MERS-CoV. In oneembodiment, broadly neutralizing antibodies to MERS-CoV S protein aredistinct from other antibodies to MERS-CoV S protein in that theyneutralize a high percentage of the many types of MERS-CoV incirculation.

Nucleic acid: A polymer composed of nucleotide units (ribonucleotides,deoxyribonucleotides, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof) linked viaphosphodiester bonds, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof. Thus, the termincludes nucleotide polymers in which the nucleotides and the linkagesbetween them include non-naturally occurring synthetic analogs, such as,for example and without limitation, phosphorothioates, phosphoramidates,methyl phosphonates, chiral-methyl phosphonates, 2-O-methylribonucleotides, peptide-nucleic acids (PNAs), and the like. Suchpolynucleotides can be synthesized, for example, using an automated DNAsynthesizer. The term “oligonucleotide” typically refers to shortpolynucleotides, generally no greater than about 50 nucleotides. It willbe understood that when a nucleotide sequence is represented by a DNAsequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e.,A, U, G, C) in which “U” replaces “T.”

“Nucleotide” includes, but is not limited to, a monomer that includes abase linked to a sugar, such as a pyrimidine, purine or syntheticanalogs thereof, or a base linked to an amino acid, as in a peptidenucleic acid (PNA). A nucleotide is one monomer in a polynucleotide. Anucleotide sequence refers to the sequence of bases in a polynucleotide.

Conventional notation is used herein to describe nucleotide sequences:the left-hand end of a single-stranded nucleotide sequence is the5′-end; the left-hand direction of a double-stranded nucleotide sequenceis referred to as the 5′-direction. The direction of 5′ to 3′ additionof nucleotides to nascent RNA transcripts is referred to as thetranscription direction. The DNA strand having the same sequence as anmRNA is referred to as the “coding strand;” sequences on the DNA strandhaving the same sequence as an mRNA transcribed from that DNA and whichare located 5′ to the 5′-end of the RNA transcript are referred to as“upstream sequences;” sequences on the DNA strand having the samesequence as the RNA and which are 3′ to the 3′ end of the coding RNAtranscript are referred to as “downstream sequences.”

“cDNA” refers to a DNA that is complementary or identical to an mRNA, ineither single stranded or double stranded form.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA produced by that geneproduces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and non-codingstrand, used as the template for transcription, of a gene or cDNA can bereferred to as encoding the protein or other product of that gene orcDNA. Unless otherwise specified, a “nucleotide sequence encoding anamino acid sequence” includes all nucleotide sequences that aredegenerate versions of each other and that encode the same amino acidsequence. Nucleotide sequences that encode proteins and RNA may includeintrons.

A first sequence is an “antisense” with respect to a second sequence ifa polynucleotide whose sequence is the first sequence specificallyhybridizes with a polynucleotide whose sequence is the second sequence.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter, such as the CMV promoter, isoperably linked to a coding sequence if the promoter affects thetranscription or expression of the coding sequence. Generally, operablylinked DNA sequences are contiguous and, where necessary to join twoprotein-coding regions, in the same reading frame.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers of use are conventional. Remington's Pharmaceutical Sciences,by E. W. Martin, Mack Publishing Co., Easton, Pa., 19th Edition, 1995,describes compositions and formulations suitable for pharmaceuticaldelivery of the disclosed immunogens.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate. In particular embodiments, suitable foradministration to a subject the carrier may be sterile, and/or suspendedor otherwise contained in a unit dosage form containing one or moremeasured doses of the composition suitable to induce the desiredanti-MERS-CoV immune response. It may also be accompanied by medicationsfor its use for treatment purposes. The unit dosage form may be, forexample, in a sealed vial that contains sterile contents or a syringefor injection into a subject, or lyophilized for subsequentsolubilization and administration or in a solid or controlled releasedosage.

Polypeptide: Any chain of amino acids, regardless of length orpost-translational modification (e.g., glycosylation orphosphorylation). “Polypeptide” applies to amino acid polymers includingnaturally occurring amino acid polymers and non-naturally occurringamino acid polymer as well as in which one or more amino acid residue isa non-natural amino acid, for example an artificial chemical mimetic ofa corresponding naturally occurring amino acid. A “residue” refers to anamino acid or amino acid mimetic incorporated in a polypeptide by anamide bond or amide bond mimetic. A polypeptide has an amino terminal(N-terminal) end and a carboxy terminal (C-terminal) end. “Polypeptide”is used interchangeably with peptide or protein, and is used herein torefer to a polymer of amino acid residues. A protein can includemultiple polypeptide chains; for example, mature MERS-CoV S proteinincludes S1 and S2 polypeptide chains.

Amino acids in a peptide, polypeptide or protein generally arechemically bound together via amide linkages (CONH). Additionally, aminoacids may be bound together by other chemical bonds. For example,linkages for amino acids or amino acid analogs can include CH₂NH—,—CH₂S—, —CH₂—CH₂ —CH═CH— (cis and trans), —COCH₂ —CH(OH)CH₂—, and—CHH₂SO— (These and others can be found in Spatola, in Chemistry andBiochemistry of Amino Acids, Peptides, and Proteins, B. Weinstein, eds.,Marcel Dekker, New York, p. 267 (1983); Spatola, A. F., Vega Data (March1983), Vol. 1, Issue 3, Peptide Backbone Modifications (general review);Morley, Trends Pharm Sci pp. 463-468, 1980; Hudson, et al., Int J PeptProt Res 14:177-185, 1979; Spatola et al. Life Sci 38:1243-1249, 1986;Harm J. Chem. Soc Perkin Trans. 1307-314, 1982; Almquist et al. J. Med.Chem. 23:1392-1398, 1980; Jennings-White et al. Tetrahedron Lett23:2533, 1982; Holladay et al. Tetrahedron. Lett 24:4401-4404, 1983; andHruby Life Sci 31:189-199, 1982.

Polypeptide modifications: Polypeptides and peptides, such as theMERS-CoV S proteins disclosed herein can be modified by a variety ofchemical techniques to produce derivatives having essentially the sameactivity as the unmodified peptides, and optionally having otherdesirable properties. For example, carboxylic acid groups of theprotein, whether carboxyl-terminal or side chain, may be provided in theform of a salt of a pharmaceutically-acceptable cation or esterified toform a C₁-C₁₆ ester, or converted to an amide of formula NR₁R₂ whereinR₁ and R₂ are each independently H or C₁-C₁₆ alkyl, or combined to forma heterocyclic ring, such as a 5- or 6-membered ring. Amino groups ofthe peptide, whether amino-terminal or side chain, may be in the form ofa pharmaceutically-acceptable acid addition salt, such as the HCl, HBr,acetic, benzoic, toluene sulfonic, maleic, tartaric and other organicsalts, or may be modified to C₁-C₁₆ alkyl or dialkyl amino or furtherconverted to an amide.

Hydroxyl groups of the peptide side chains can be converted to C₁-C₁₆alkoxy or to a C₁-C₁₆ ester using well-recognized techniques. Phenyl andphenolic rings of the peptide side chains can be substituted with one ormore halogen atoms, such as F, Cl, Br or I, or with C₁-C₁₆ alkyl, C₁-C₁₆alkoxy, carboxylic acids and esters thereof, or amides of suchcarboxylic acids. Methylene groups of the peptide side chains can beextended to homologous C₂-C₄ alkylenes. Thiols can be protected with anyone of a number of well-recognized protecting groups, such as acetamidegroups. Those skilled in the art will also recognize methods forintroducing cyclic structures into the peptides of this disclosure toselect and provide conformational constraints to the structure thatresult in enhanced stability. For example, a C- or N-terminal cysteinecan be added to the peptide, so that when oxidized the peptide willcontain a disulfide bond, generating a cyclic peptide. Other peptidecyclizing methods include the formation of thioethers and carboxyl- andamino-terminal amides and esters.

Prime-boost vaccination: An immunotherapy including administration to asubject of a first immunogenic composition (the primer vaccine)including a selected target antigen or nucleic acid molecule encodingthe target antigen, followed by administration of a second immunogeniccomposition (the booster vaccine) including the target antigen ornucleic acid molecule encoding the target antigen, to induce an immuneresponse to the target antigen in the subject. The booster vaccine isadministered to the subject after the primer vaccine; the skilledartisan will understand a suitable time interval between administrationof the primer vaccine and the booster vaccine, and examples of suchtimeframes are disclosed herein. Additional administrations can beincluded in the prime-boost protocol, for example a second boost. Insome embodiments, the primer vaccine, the booster vaccine, or bothprimer vaccine and the booster vaccine additionally include an adjuvant.In one non-limiting example, the primer vaccine is a DNA-based vaccine(or other vaccine based on gene delivery), and the booster vaccine is aprotein-based vaccine (such as a protein subunit or proteinnanoparticle).

Protein nanoparticle: A multi-subunit, protein-based polyhedron shapedstructure. The subunits are each composed of proteins or polypeptides(for example a glycosylated polypeptide), and, optionally of single ormultiple features of the following: nucleic acids, prosthetic groups,organic and inorganic compounds. Non-limiting examples of proteinnanoparticles include ferritin nanoparticles (see, e.g., Zhang, Y. Int.J. Mol. Sci., 12:5406-5421, 2011, incorporated by reference herein),encapsulin nanoparticles (see, e.g., Sutter et al., Nature Struct. andMol. Biol., 15:939-947, 2008, incorporated by reference herein), SulfurOxygenase Reductase (SOR) nanoparticles (see, e.g., Urich et al.,Science, 311:996-1000, 2006, incorporated by reference herein), lumazinesynthase nanoparticles (see, e.g., Zhang et al., J. Mol. Biol., 306:1099-1114, 2001) or pyruvate dehydrogenase nanoparticles (see, e.g.,Izard et al., PNAS 96: 1240-1245, 1999, incorporated by referenceherein). Ferritin, encapsulin, SOR, lumazine synthase, and pyruvatedehydrogenase are monomeric proteins that self-assemble into a globularprotein complexes that in some cases consists of 24, 60, 24, 60, and 60protein subunits, respectively. In some examples, ferritin, encapsulin,SOR, lumazine synthase, or pyruvate dehydrogenase monomers are linked toa MERS-CoV S protein (or fragment thereof) and self-assemble into aprotein nanoparticle presenting the MERS-CoV S protein on its surface,which can be administered to a subject to stimulate an immune responseto the antigen.

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination can be accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids, for example, by genetic engineering techniques. Arecombinant protein is one that has a sequence that is not naturallyoccurring or has a sequence that is made by an artificial combination oftwo otherwise separated segments of sequence. In several embodiments, arecombinant protein is encoded by a heterologous (for example,recombinant) nucleic acid that has been introduced into a host cell,such as a bacterial or eukaryotic cell. The nucleic acid can beintroduced, for example, on an expression vector having signals capableof expressing the protein encoded by the introduced nucleic acid or thenucleic acid can be integrated into the host cell chromosome.

Sample (or biological sample): A biological specimen containing genomicDNA, RNA (including mRNA), protein, or combinations thereof, obtainedfrom a subject. Examples include, but are not limited to, peripheralblood, tissue, cells, urine, saliva, tissue biopsy, fine needleaspirate, surgical specimen, and autopsy material.

Sequence identity: The similarity between amino acid sequences isexpressed in terms of the similarity between the sequences, otherwisereferred to as sequence identity. Sequence identity is frequentlymeasured in terms of percentage identity (or similarity or homology);the higher the percentage, the more similar the two sequences are.Homologs, orthologs, or variants of a polypeptide will possess arelatively high degree of sequence identity when aligned using standardmethods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smith &Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol.Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp,CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988;Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; andPearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J.Mol. Biol. 215:403-10, 1990, presents a detailed consideration ofsequence alignment methods and homology calculations.

Once aligned, the number of matches is determined by counting the numberof positions where an identical nucleotide or amino acid residue ispresent in both sequences. The percent sequence identity is determinedby dividing the number of matches either by the length of the sequenceset forth in the identified sequence, or by an articulated length (suchas 100 consecutive nucleotides or amino acid residues from a sequenceset forth in an identified sequence), followed by multiplying theresulting value by 100. For example, a peptide sequence that has 1166matches when aligned with a test sequence having 1554 amino acids is75.0 percent identical to the test sequence (1166÷1554*100=75.0). Thepercent sequence identity value is rounded to the nearest tenth. Forexample, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The lengthvalue will always be an integer.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403, 1990) is available from several sources, includingthe National Center for Biotechnology Information (NCBI, Bethesda, Md.)and on the internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. A description ofhow to determine sequence identity using this program is available onthe NCBI website on the internet.

Homologs and variants of a polypeptide are typically characterized bypossession of at least about 75%, for example at least about 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identitycounted over the full length alignment with the amino acid sequence ofinterest. Proteins with even greater similarity to the referencesequences will show increasing percentage identities when assessed bythis method, such as at least 80%, at least 85%, at least 90%, at least95%, at least 98%, or at least 99% sequence identity. When less than theentire sequence is being compared for sequence identity, homologs andvariants will typically possess at least 80% sequence identity overshort windows of 10-20 amino acids, and may possess sequence identitiesof at least 85% or at least 90% or 95% depending on their similarity tothe reference sequence. Methods for determining sequence identity oversuch short windows are available at the NCBI website on the internet.One of skill in the art will appreciate that these sequence identityranges are provided for guidance only; it is entirely possible thatstrongly significant homologs could be obtained that fall outside of theranges provided.

For sequence comparison of nucleic acid sequences, typically onesequence acts as a reference sequence, to which test sequences arecompared. When using a sequence comparison algorithm, test and referencesequences are entered into a computer, subsequence coordinates aredesignated, if necessary, and sequence algorithm program parameters aredesignated. Default program parameters are used. Methods of alignment ofsequences for comparison are well known in the art. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith & Waterman, Adv. Appl. Math. 2:482, 1981, by thehomology alignment algorithm of Needleman & Wunsch, J. Mol. Biol.48:443, 1970, by the search for similarity method of Pearson & Lipman,Proc. Nat'l. Acad. Sci. USA 85:2444, 1988, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by manual alignment and visualinspection (see, e.g., Sambrook et al. (Molecular Cloning: A LaboratoryManual, 4^(th) ed, Cold Spring Harbor, N.Y., 2012) and Ausubel et al.(In Current Protocols in Molecular Biology, John Wiley & Sons, New York,through supplement 104, 2013). One example of a useful algorithm isPILEUP. PILEUP uses a simplification of the progressive alignment methodof Feng & Doolittle, J. Mol. Evol. 35:351-360, 1987. The method used issimilar to the method described by Higgins & Sharp, CABIOS 5:151-153,1989. Using PILEUP, a reference sequence is compared to other testsequences to determine the percent sequence identity relationship usingthe following parameters: default gap weight (3.00), default gap lengthweight (0.10), and weighted end gaps. PILEUP can be obtained from theGCG sequence analysis software package, e.g., version 7.0 (Devereaux etal., Nuc. Acids Res. 12:387-395, 1984.

Another example of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and the BLAST2.0 algorithm, which are described in Altschul et al., J. Mol. Biol.215:403-410, 1990 and Altschul et al., Nucleic Acids Res. 25:3389-3402,1977. Software for performing BLAST analyses is publicly availablethrough the National Center for Biotechnology Information(ncbi.nlm.nih.gov). The BLASTN program (for nucleotide sequences) usesas defaults a word length (W) of 11, alignments (B) of 50, expectation(E) of 10, M=5, N=−4, and a comparison of both strands. The BLASTPprogram (for amino acid sequences) uses as defaults a word length (W) of3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989). Anoligonucleotide is a linear polynucleotide sequence of up to about 100nucleotide bases in length.

As used herein, reference to “at least 80% identity” refers to “at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or even 100% identity” to a specified referencesequence.

Signal Peptide: A short amino acid sequence (e.g., approximately 10-35amino acids in length) that directs newly synthesized secretory ormembrane proteins to and through membranes (for example, the endoplasmicreticulum membrane). Signal peptides are typically located at theN-terminus of a polypeptide and are removed by signal peptidases. Signalpeptide sequences typically contain three common structural features: anN-terminal polar basic region (n-region), a hydrophobic core, and ahydrophilic c-region). Exemplary signal peptide sequences are set forthas residues 1-18 of SEQ ID NO: 14 (MERS-CoV S protein England1 strain).

Specifically bind: When referring to the formation of anantibody:antigen protein complex, or a protein:protein complex, refersto a binding reaction which determines the presence of a target protein,peptide, or polysaccharide (for example a glycoprotein), in the presenceof a heterogeneous population of proteins and other biologics. Thus,under designated conditions, an particular antibody or protein bindspreferentially to a particular target protein, peptide or polysaccharide(such as an antigen present on the surface of a pathogen, for exampleMERS-CoV S protein) and does not bind in a significant amount to otherproteins or polysaccharides present in the sample or subject. Specificbinding can be determined by methods known in the art. A first proteinor antibody “specifically binds to a target protein when the interactionhas a K_(D) of less than about 10⁻⁶ Molar, such as less than about 10⁻⁷Molar, 10⁻⁸ Molar, 10, or even less than about 10⁻¹⁰ Molar.

A variety of immunoassay formats are appropriate for selectingantibodies or other ligands specifically immunoreactive with aparticular protein. For example, solid-phase ELISA immunoassays areroutinely used to select monoclonal antibodies specificallyimmunoreactive with a protein. See Harlow & Lane, Antibodies, ALaboratory Manual, 2^(nd) ed., Cold Spring Harbor Publications, New York(2013), for a description of immunoassay formats and conditions that canbe used to determine specific immunoreactivity.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes human and non-human mammals. In an example, a subject is ahuman. In a particular example, the subject is a human or a camel, or abat. In an additional example, a subject is selected that is in need ofinhibiting of an MERS-CoV infection. For example, the subject is eitheruninfected and at risk of MERS-CoV infection or is infected and in needof treatment.

Therapeutically effective amount: The amount of agent, such as adisclosed immunogen or antibody, that is sufficient to prevent, treat(including prophylaxis), reduce and/or ameliorate the symptoms and/orunderlying causes of a disorder or disease, for example to prevent,inhibit, and/or treat MERS-CoV infection. In some embodiments, atherapeutically effective amount is sufficient to reduce or eliminate asymptom of a disease, such as MERS-CoV infection. For instance, this canbe the amount necessary to inhibit or prevent viral replication or tomeasurably alter outward symptoms of the viral infection. In general,this amount will be sufficient to measurably inhibit virus replicationor infectivity.

In one example, a desired response is to inhibit or reduce or preventMERS-CoV infection. The MERS-CoV infected cells do not need to becompletely eliminated or reduced or prevented for the composition to beeffective. For example, administration of a therapeutically effectiveamount of the agent can decrease the number of MERS-CoV infected cells(or prevent the infection of cells) by a desired amount, for example byat least 10%, at least 20%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, at least 98%, or even at least100% (elimination or prevention of detectable MERS-CoV infected cells),as compared to the number of MERS-CoV infected cells in the absence ofthe composition.

It is understood that to obtain a protective immune response against apathogen can require multiple administrations of a disclosed immunogen.Thus, a therapeutically effective amount encompasses a fractional dosethat contributes in combination with previous or subsequentadministrations to attaining a desired immune response. For example, atherapeutically effective amount of an immunogen can be administered ina single dose, or in several doses, for example daily, during a courseof treatment (such as a prime-boost vaccination treatment).

The therapeutically effective amount of a disclosed immunogen orantibody can depend on the subject being treated, the severity and typeof the condition being treated, and the manner of administration. A unitdosage form of the immunogen or antibody can be packaged in atherapeutic amount, or in multiples of the therapeutic amount, forexample, in a vial (e.g., with a pierceable lid) or syringe havingsterile components.

Transmembrane domain: An amino acid sequence that inserts into a lipidbilayer, such as the lipid bilayer of a cell or virus or virus-likeparticle. A transmembrane domain can be used to anchor an antigen to amembrane. In some examples a transmembrane domain is a MERS-CoV Sprotein transmembrane domain. Exemplary MERS-CoV S protein transmembranedomains are familiar to the person of ordinary skill in the art, andprovided herein, for example as residues 1296-1318 of SEQ ID NO: 14.

Treating or preventing a disease: Inhibiting the full development of adisease or condition, for example, in a subject who is at risk for adisease such as MERS-CoV infection. “Treatment” refers to a therapeuticintervention that ameliorates a sign or symptom of a disease orpathological condition after it has begun to develop. The term“ameliorating,” with reference to a disease or pathological condition,refers to any observable beneficial effect of the treatment. Thebeneficial effect can be evidenced, for example, by a delayed onset ofclinical symptoms of the disease in a susceptible subject, a reductionin severity of some or all clinical symptoms of the disease, a slowerprogression of the disease, a reduction in the viral load, animprovement in the overall health or well-being of the subject, or byother parameters well known in the art that are specific to theparticular disease. A “prophylactic” treatment is a treatmentadministered to a subject who does not exhibit signs of a disease orexhibits only early signs for the purpose of decreasing the risk ofdeveloping pathology.

The term “reduces” is a relative term, such that an agent reduces aresponse or condition if the response or condition is quantitativelydiminished following administration of the agent, or if it is diminishedfollowing administration of the agent, as compared to a reference agent.Similarly, the term “prevents” does not necessarily mean that an agentcompletely eliminates the response or condition, so long as at least onecharacteristic of the response or condition is eliminated. Thus, animmunogenic composition that reduces or prevents an infection or aresponse, can, but does not necessarily completely, eliminate such aninfection or response, so long as the infection or response ismeasurably diminished, for example, by at least about 50%, such as by atleast about 70%, or about 80%, or even by about 90% of (that is to 10%or less than) the infection or response in the absence of the agent, orin comparison to a reference agent.

Under conditions sufficient for: A phrase that is used to describe anyenvironment that permits a desired activity.

Vaccine: A pharmaceutical composition that induces a prophylactic ortherapeutic immune response in a subject. In some cases, the immuneresponse is a protective immune response. Typically, a vaccine inducesan antigen-specific immune response to an antigen of a pathogen, forexample a viral pathogen, or to a cellular constituent correlated with apathological condition. A vaccine may include a polynucleotide (such asa nucleic acid encoding a disclosed antigen), a peptide or polypeptide(such as a disclosed antigen), a virus, a cell or one or more cellularconstituents. In one specific, non-limiting example, a vaccine inducesan immune response that reduces the severity of the symptoms associatedwith MERS-CoV infection and/or decreases the viral load compared to acontrol. In another non-limiting example, a vaccine induces an immuneresponse that reduces and/or prevents MERS-CoV infection compared to acontrol.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. Recombinant DNA vectors are vectorshaving recombinant DNA. A vector can include nucleic acid sequences thatpermit it to replicate in a host cell, such as an origin of replication.A vector can also include one or more selectable marker genes and othergenetic elements known in the art. Viral vectors are recombinant nucleicacid vectors having at least some nucleic acid sequences derived fromone or more viruses. A replication deficient viral vector is a vectorthat requires complementation of one or more regions of the viral genomerequired for replication due to a deficiency in at least onereplication-essential gene function. For example, such that the viralvector does not replicate in typical host cells, especially those in ahuman patient that could be infected by the viral vector in the courseof a therapeutic method.

Virus-like particle (VLP): A non-replicating, viral shell, derived fromany of several viruses. VLPs are generally composed of one or more viralproteins, such as, but not limited to, those proteins referred to ascapsid, coat, shell, surface and/or envelope proteins, orparticle-forming polypeptides derived from these proteins. VLPs can formspontaneously upon recombinant expression of the protein in anappropriate expression system. Methods for producing particular VLPs areknown in the art. The presence of VLPs following recombinant expressionof viral proteins can be detected using conventional techniques known inthe art, such as by electron microscopy, biophysical characterization,and the like. Further, VLPs can be isolated by known techniques, e.g.,density gradient centrifugation and identified by characteristic densitybanding. See, for example, Baker et al. (1991) Biophys. J. 60:1445-1456;and Hagensee et al. (1994) J. Virol. 68:4503-4505; Vincente, J InvertebrPathol., 2011; Schneider-Ohrum and Ross, Curr. Top. Microbiol. Immunol.,354: 53073, 2012).

II. Neutralizing Monoclonal Antibodies and their Use

Isolated monoclonal antibodies and antigen binding fragments thereofthat specifically bind an epitope on MERS-CoV S protein are provided.The antibodies and antigen binding fragments can be humanized. Inseveral embodiments, the antibodies and antigen binding fragments can beused to inhibit or treat MERS-CoV infection. Also disclosed herein arecompositions including the antibodies and antigen binding fragments anda pharmaceutically acceptable carrier. Nucleic acids encoding theantibodies or antigen binding fragments, expression vectors includingthese nucleic acids, and isolated host cells that express the nucleicacids are also provided.

The antibodies, antigen binding fragments, nucleic acid molecules, hostcells, and compositions can be used for research, diagnostic andtherapeutic purposes. For example, the monoclonal antibodies and antigenbinding fragments can be used to diagnose or treat a subject with aMERS-CoV infection. Additionally, the antibodies can be used todetermine MERS-CoV titer in a subject. The antibodies disclosed hereinalso can be used to study the biology of MERS-CoV.

A. Antibodies and Antigen Binding Fragments

Isolated monoclonal antibodies and antigen binding fragments thatspecifically bind an epitope on MERS-CoV S protein are provided. Theantibodies and antigen binding fragments can neutralize MERS-CoVinfection.

In some embodiments, the antibodies and antigen binding fragmentsinclude a variable heavy (V_(H)) and a variable light (V_(L)) chain andspecifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In several embodiments, the antibodies and antigen bindingfragments include a heavy chain comprising a heavy chain complementaritydetermining region (HCDR)₁, a HCDR2 and an HCDR3, and a light chaincomprising a light chain complementarity determining region (LCDR) 1, aLCDR2, and a LCDR3 and specifically bind to MERS-CoV S protein andoptionally also neutralize MERS-CoV infection. In several embodiments,the antibody or antigen binding fragment includes heavy and light chainvariable regions including the HCDR1, HCDR2, and HCDR3, and LCDR1,LCDR2, and LCDR3, respectively, of one of the JC57-13, JC57-14, JC57-11,C2, C5, A2, A10, FIB_B2, FIB_H1, G2, G4, D12, or F11 antibodies, andspecifically binds to MERS-CoV S protein and optionally also neutralizesMERS-CoV infection.

The discussion of monoclonal antibodies below refers to isolatedmonoclonal antibodies that include heavy and/or light chain variabledomains (or antigen binding fragments thereof) including a CDR1, CDR2,and/or CDR3 with reference to the IMGT numbering scheme (unless thecontext indicates otherwise). The person of ordinary skill in the artwill understand that various CDR numbering schemes (such as the Kabat,Chothia or IMGT numbering schemes) can be used to determine CDRpositions. The amino acid sequence and the CDR positions of the heavyand light chain of the JC57-13, JC57-14, JC57-11, C2, C5, A2, A10,FIB_B2, FIB_H1, G2, G4, D12, and F11 monoclonal antibodies according tothe IMGT numbering schemes are shown in Table 1 (IMGT).

TABLE 1 IMGT CDR sequences of MERS-CoV S protein specific antibodiesJC57-13 V_(H) CDR SEQ ID NO: 2 SEQ V_(H) residues A.A. Sequence ID NOHCDR1 26-33 GGSISSNY 59 HCDR2 51-58 IYGGSGST 60 HCDR3  97-110ARLLPLGGGYCFDY 61 JC57-13 V_(L) CDR SEQ ID NO: 4 SEQ V_(L) residues A.A.Sequence ID NO LCDR1 27-38 QSLFDSDYGNTY 62 LCDR2 56-58 MLS 63 LCDR3 95-103 MQSVEYPFT 64 JC57-11 V_(H) CDR SEQ ID NO: 6 SEQ V_(H) residuesA.A. Sequence ID NO HCDR1 27-34 GSISDSYR 65 HCDR2 52-59 IFATGTTT 66HCDR3  98-120 AREPFKYCSGGVCYAHKDNSLDV 67 JC57-11 V_(L) CDR SEQ ID NO: 8SEQ V_(L) residues A.A. Sequence ID NO LCDR1 27-32 QSVSSN 68 LCDR2 50-52SAS 69 LCDR3 89-96 YQHSSGYT 70 JC57-14 V_(H) CDR SEQ ID NO: 10 SEQ V_(H)residues A.A. Sequence ID NO HCDR1 26-33 GDSISSNY 71 HCDR2 51-58FSGSGGST 72 HCDR3  97-106 AKTYSGTFDY 73 JC57-14 V_(L) CDR SEQ ID NO: 12SEQ V_(L) residues A.A. Sequence ID NO LCDR1 27-32 QDINNY 74 LCDR2 50-52YAS 75 LCDR3 89-97 QQYNNSPYS 76 C2 V_(H) CDR SEQ ID NO: 36 SEQ V_(H)Residues A.A. Sequence ID NO HCDR1 26-33 GGTFSIYA 77 HCDR2 51-58IIPIFGTA 78 HCDR3  97-116 AREGGHQGYCSGGSCYDFDY 79 C2 V_(L) CDR SEQ IDNO: 38 SEQ V_(L) Residues A.A. Sequence ID NO LCDR1 27-37 QSLLHSNGYNY 80LCDR2 55-57 LGS 81 LCDR3  94-101 MQALQTPA 82 C2 LCDR1 NG-NS V_(L) SEQ IDNO: CDR 110 SEQ V_(L) Residues A.A. Sequence ID NO LCDR1 27-37QSLLHSNSYNY 112  LCDR2 55-57 LGS 81 LCDR3  94-101 MQALQTPA 82 C2 LCDR1NG-NA V_(L) SEQ ID NO: CDR 111 SEQ V_(L) Residues A.A. Sequence ID NOLCDR1 27-37 QSLLHSNAYNY 113  LCDR2 55-57 LGS 81 LCDR3  94-101 MQALQTPA82 C5 V_(H) CDR SEQ ID NO: 40 SEQ V_(H) Residues A.A. Sequence ID NOHCDR1 26-35 GGSISSSSYY 83 HCDR2 53-59 IYYSGST 84 HCDR3  98-115ASLLRPLIYCSGGSCTDY 85 C5 V_(L) CDR SEQ ID NO: 42 SEQ V_(L) Residues A.A.Sequence ID NO LCDR1 26-34 SSDVGGYNY 86 LCDR2 52-54 EVS 87 LCDR3  91-100SSYTSNITLV 88 A2 V_(H) CDR SEQ ID NO: 44 SEQ V_(H) Residues A.A.Sequence ID NO HCDR1 26-33 GFTFSDYY 89 HCDR2 51-58 ISSSGSTI 90 HCDR3 97-111 ARVGLGSGWYDWFDP 91 A2 V_(L) CDR SEQ ID NO: 46 SEQ V_(L) ResiduesA.A. Sequence ID NO LCDR1 26-34 SSNIGASYD 92 LCDR2 52-54 GNT 93 LCDR3 91-101 QSYDSSLSGVV 94 A10 V_(H) CDR SEQ ID NO: 48 SEQ V_(H) ResiduesA.A. Sequence ID NO HCDR1 26-33 GGTFSTYA 95 HCDR2 51-58 IIPIFGTA 78HCDR3  97-111 ARGSRSSSSAEYFQH 96 A10 V_(L) CDR SEQ ID NO: 50 SEQ V_(L)Residues A.A. Sequence ID NO LCDR1 26-34 SSDVGGYNY 86 LCDR2 52-54 DVS 97LCDR3  92-102 SYAGSYTLEVV 98 FIB_B2 V_(H) CDR SEQ ID NO: 52 SEQ V_(H)Residues A.A. Sequence ID NO HCDR1 26-33 GGSISSNY 59 HCDR2 51-55IYGGSGGT 99 HCDR3  97-107 ARSFYSWNGES 100  FIB_B2 V_(L) CDR SEQ ID NO:54 SEQ V_(L) Residues A.A. Sequence ID NO LCDR1 27-32 QGINDY 101 LCDR250-52 YGN 102 LCDR3 89-97 QQGDSFPLT 103 FIB_H1 V_(H) CDR SEQ ID NO: 56SEQ V_(H) Residues A.A. Sequence ID NO HCDR1 26-33 GHIFTSYV 104 HCDR251-58 IHPGNGGR 105 HCDR3  97-110 AASSGSYGVSSLDV 106 FIB_H1 V_(L) CDR SEQID NO: 58 SEQ V_(L) Residues A.A. Sequence ID NO LCDR1 26-34 SDLSVGSKN107 LCDR2 52-58 YYSDSDK 108 LCDR3  97-106 QVYDSSANWV 109 G2 V_(H) SEQ IDNO: CDR 115 SEQ V_(H) Residues A.A. Sequence ID NO HCDR1 26-33 GFTFSSSY130 HCDR2 51-58 IYAGTGGT 131 HCDR3  97-107 ARGGSSFAMDY 132 G2 V_(L) SEQID NO: CDR 117 SEQ V_(L) Residues A.A. Sequence ID NO LCDR1 26-36ESVDNYGISF 133 LCDR2 54-56 TAS 134 LCDR3 93-97 QQSEE 135 G4 V_(H) SEQ IDNO: CDR 119 SEQ V_(H) Residues A.A. Sequence ID NO HCDR1 26-33 GYTFTDYA136 HCDR2 51-58 FSTYYGNT 137 HCDR3  97-111 ARKSYYVDYVDAMDY 138 G4 V_(L)SEQ ID NO: CDR 121 SEQ V_(L) Residues A.A. Sequence ID NO LCDR1 26-36ESVDNYGISF 139 LCDR2 54-56 ATS 140 LCDR3  93-101 QQSKEVPRT 141 D12 V_(H)SEQ ID NO: CDR 123 SEQ V_(H) Residues A.A. Sequence ID NO HCDR1 26-33GFTFSSYA 142 HCDR2 51-58 ISSGGTYT 143 HCDR3  97-105 VRDGNSMDY 144 D12V_(L) SEQ ID NO: CDR 125 SEQ V_(L) Residues A.A. Sequence ID NO LCDR126-32 QDINNY  74 LCDR2 50-52 YTS 145 LCDR3 89-97 QQANTLPPT 146 F11 V_(H)SEQ ID NO: CDR 127 SEQ V_(H) Residues A.A. Sequence ID NO HCDR1 26-33GFTFSRYA 147 HCDR2 51-58 INNGGSYS 148 HCDR3  97-111 ARHYDYDGYYYTMDF 149F11 V_(L) SEQ ID NO: CDR 129 SEQ V_(L) Residues A.A. Sequence ID NOLCDR1 26-37 QSIVHSNGNTY 150 LCDR2 55-57 KVS 151 LCDR3  94-102 FQGSHVPYT152

JC57-13

In some embodiments, the antibody or antigen binding fragment can bebased on or derived from the JC57-13 antibody, and can specifically bindto MERS-CoV S protein and neutralize MERS-CoV infection. For example,the antibody or antigen binding fragment can comprise a V_(H) and aV_(L) comprising the HCDR1, the HCDR2, and the HCDR3, and the LCDR1, theLCDR2, and the LCDR3, respectively (for example, according to IMGT orkabat), of the JC57-13 antibody, and can specifically bind to MERS-CoV Sprotein and neutralize MERS-CoV infection.

In some embodiments, the antibody or antigen binding fragment cancomprise a V_(H) comprising the HCDR1, the HCDR2, and the HCDR3 of theJC57-13 V_(H) as set forth in Table 1, and can specifically bind toMERS-CoV S protein and neutralize MERS-CoV infection. In someembodiments, the antibody or antigen binding fragment can comprise aV_(L) comprising the LCDR1, the LCDR2, and the LCDR3 of the JC57-13V_(L) as set forth in Table 1, and can specifically bind to MERS-CoV Sprotein and neutralize MERS-CoV infection. In some embodiments, theantibody or antigen binding fragment can comprise a V_(H) and a V_(L)comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, andthe LCDR3 of the JC57-13 V_(H) and V_(L) as set forth in Table 1, andcan specifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection.

In some embodiments, the antibody or antigen binding fragment includesat least one CDR (such as an HCDR3) with a sequence that has at least95% (such as at least 96%, at least 97%, at least 98%, at least 99%, oreven 100%) sequence identity to any one of the heavy or light chain CDRsof the JC57-13 V_(H) or V_(L) as shown in Table 1, and can specificallybind to MERS-CoV S protein and neutralize MERS-CoV infection. In someembodiments, the antibody or antigen binding fragment includes a V_(H)comprising a HCDR1, a HCDR2, and a HCDR3 comprising amino acid sequencesat least 90% (such as at least 95%, at least 96%, at least 97%, at least98%, or at least 99%) identical to amino acids 26-33, 51-58, and 97-110,respectively, of SEQ ID NO: 2, and can specifically bind to MERS-CoV Sprotein and neutralize MERS-CoV infection. In some embodiments, theantibody or antigen binding fragment includes a V_(L) comprising aLCDR1, a LCDR2, and a LCDR3 comprising amino acid sequences at least 90%(such as at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%) identical to amino acids amino acids 27-38, 56-58, and95-103, respectively, of SEQ ID NO: 4, and can specifically bind toMERS-CoV S protein and neutralize MERS-CoV infection. In additionalembodiments, the antibody or antigen binding fragment includes a V_(H)comprising a HCDR1, a HCDR2, and a HCDR3 comprising amino acid sequencesat least 90% (such as at least 95%, at least 96%, at least 97%, at least98%, or at least 99%) identical to amino acids 26-33, 51-58, and 97-110,respectively, of SEQ ID NO: 2, and a V_(L) comprising a LCDR1, a LCDR2,and a LCDR3 comprising amino acid sequences at least 90% (such as atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to amino acids amino acids 27-38, 56-58, and 95-103,respectively, of SEQ ID NO: 2, and can specifically bind to MERS-CoV Sprotein and neutralize MERS-CoV infection.

In some embodiments, the antibody or antigen binding fragment includes aV_(H) comprising an amino acid sequence at least 90% (such as at least95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to the amino acid sequence set forth as SEQ ID NO: 2, and canspecifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In more embodiments, the antibody or antigen binding fragmentincludes a V_(L) comprising an amino acid sequence at least 90% (such asat least 95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to the amino acid sequence set forth as SEQ ID NO: 4, and canspecifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In additional embodiments, the antibody or antigen bindingfragment includes a V_(H) and a V_(L) independently comprising aminoacid sequences at least 90% (such as at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%) identical to the amino acidsequences set forth as SEQ ID NOs: 2 and 4, respectively, and canspecifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection.

In additional embodiments, the antibody or antigen binding fragmentincludes a V_(H) comprising the amino acid sequence set forth as one ofSEQ ID NO: 2, and can specifically bind to MERS-CoV S protein andneutralize MERS-CoV infection. In more embodiments, the antibody orantigen binding fragment includes a V_(L) comprising the amino acidsequence set forth as SEQ ID NO: 4, and can specifically bind toMERS-CoV S protein and neutralize MERS-CoV infection. In someembodiments, the antibody or antigen binding fragment includes a V_(H)and a V_(L) comprising the amino acid sequences set forth as SEQ ID NOs:2 and 4, respectively, and can specifically bind to MERS-CoV S proteinand neutralize MERS-CoV infection.

JC57-11

In some embodiments, the antibody or antigen binding fragment can bebased on or derived from the JC57-11 antibody. For example, the antibodyor antigen binding fragment can comprise a V_(H) and a V_(L) comprisingthe HCDR1, the HCDR2, and the HCDR3, and the LCDR1, the LCDR2, and theLCDR3, respectively (for example, according to IMGT), of the JC57-11antibody, and can specifically bind to MERS-CoV S protein and neutralizeMERS-CoV infection.

In some embodiments, the antibody or antigen binding fragment cancomprise a V_(H) comprising the HCDR1, the HCDR2, and the HCDR3 of theJC57-11 V_(H) as set forth in Table 1, and can specifically bind toMERS-CoV S protein and neutralize MERS-CoV infection. In someembodiments, the antibody or antigen binding fragment can comprise aV_(L) comprising the LCDR1, the LCDR2, and the LCDR3 of the JC57-11V_(L) as set forth in Table 1, and can specifically bind to MERS-CoV Sprotein and neutralize MERS-CoV infection. In some embodiments, theantibody or antigen binding fragment can comprise a V_(H) and a V_(L)comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, andthe LCDR3 of the JC57-11 V_(H) and V_(L) as set forth in Table 1, andcan specifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection.

In some embodiments, the antibody or antigen binding fragment includesat least one CDR (such as an HCDR3) with a sequence that has at least95% (such as at least 96%, at least 97%, at least 98%, at least 99%, oreven 100%) sequence identity to any one of the heavy or light chain CDRsof the JC57-11 V_(H) or V_(L) as shown in Table 1, and can specificallybind to MERS-CoV S protein and neutralize MERS-CoV infection. In someembodiments, the antibody or antigen binding fragment includes a V_(H)comprising a HCDR1, a HCDR2, and a HCDR3 comprising amino acid sequencesat least 90% (such as at least 95%, at least 96%, at least 97%, at least98%, or at least 99%) identical to amino acids 27-38, 52-59, and 98-120,respectively, of SEQ ID NO: 6, and can specifically bind to MERS-CoV Sprotein and neutralize MERS-CoV infection. In some embodiments, theantibody or antigen binding fragment includes a V_(L) comprising aLCDR1, a LCDR2, and a LCDR3 comprising amino acid sequences at least 90%(such as at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%) identical to amino acids amino acids 27-32, 50-52, and 89-96,respectively, of SEQ ID NO: 8, and can specifically bind to MERS-CoV Sprotein and neutralize MERS-CoV infection. In additional embodiments,the antibody or antigen binding fragment includes a V_(H) comprising aHCDR1, a HCDR2, and a HCDR3 comprising amino acid sequences at least 90%(such as at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%) identical to amino acids 27-38, 52-59, and 98-120,respectively, of SEQ ID NO: 6, and a V_(L) comprising a LCDR1, a LCDR2,and a LCDR3 comprising amino acid sequences at least 90% (such as atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to amino acids amino acids 27-32, 50-52, and 89-96,respectively, of SEQ ID NO: 8, and can specifically bind to MERS-CoV Sprotein and neutralize MERS-CoV infection.

In some embodiments, the antibody or antigen binding fragment includes aV_(H) comprising an amino acid sequence at least 90% (such as at least95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to the amino acid sequence set forth as SEQ ID NO: 6, and canspecifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In more embodiments, the antibody or antigen binding fragmentincludes a V_(L) comprising an amino acid sequence at least 90% (such asat least 95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to the amino acid sequence set forth as SEQ ID NO: 8, and canspecifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In additional embodiments, the antibody or antigen bindingfragment includes a V_(H) and a V_(L) independently comprising aminoacid sequences at least 90% (such as at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%) identical to the amino acidsequences set forth as SEQ ID NOs: 6 and 8, respectively, and canspecifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection.

In additional embodiments, the antibody or antigen binding fragmentincludes a V_(H) comprising the amino acid sequence set forth as one ofSEQ ID NO: 6, and can specifically bind to MERS-CoV S protein andneutralize MERS-CoV infection. In more embodiments, the antibody orantigen binding fragment includes a V_(L) comprising the amino acidsequence set forth as SEQ ID NO: 8, and can specifically bind toMERS-CoV S protein and neutralize MERS-CoV infection. In someembodiments, the antibody or antigen binding fragment includes a V_(H)and a V_(L) comprising the amino acid sequences set forth as SEQ ID NOs:6 and 8, respectively, and can specifically bind to MERS-CoV S proteinand neutralize MERS-CoV infection.

JC57-14

In some embodiments, the antibody or antigen binding fragment can bebased on or derived from the JC57-14 antibody. For example, the antibodyor antigen binding fragment can comprise a V_(H) and a V_(L) comprisingthe HCDR1, the HCDR2, and the HCDR3, and the LCDR1, the LCDR2, and theLCDR3, respectively (for example, according to IMGT), of the JC57-14antibody, and can specifically bind to MERS-CoV S protein and neutralizeMERS-CoV infection.

In some embodiments, the antibody or antigen binding fragment cancomprise a V_(H) comprising the HCDR1, the HCDR2, and the HCDR3 of theJC57-14 V_(H) as set forth in Table 1, and can specifically bind toMERS-CoV S protein and neutralize MERS-CoV infection. In someembodiments, the antibody or antigen binding fragment can comprise aV_(L) comprising the LCDR1, the LCDR2, and the LCDR3 of the JC57-14V_(L) as set forth in Table 1, and can specifically bind to MERS-CoV Sprotein and neutralize MERS-CoV infection. In some embodiments, theantibody or antigen binding fragment can comprise a V_(H) and a V_(L)comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, andthe LCDR3 of the JC57-14 V_(H) and V_(L) as set forth in Table 1, andcan specifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection.

In some embodiments, the antibody or antigen binding fragment includesat least one CDR (such as an HCDR3) with a sequence that has at least95% (such as at least 96%, at least 97%, at least 98%, at least 99%, oreven 100%) sequence identity to any one of the heavy or light chain CDRsof the JC57-14 V_(H) or V_(L) as shown in Table 1, and can specificallybind to MERS-CoV S protein and neutralize MERS-CoV infection. In someembodiments, the antibody or antigen binding fragment includes a V_(H)comprising a HCDR1, a HCDR2, and a HCDR3 comprising amino acid sequencesat least 90% (such as at least 95%, at least 96%, at least 97%, at least98%, or at least 99%) identical to amino acids 26-33, 51-58, and 97-106,respectively, of SEQ ID NO: 10, and can specifically bind to MERS-CoV Sprotein and neutralize MERS-CoV infection. In some embodiments, theantibody or antigen binding fragment includes a V_(L) comprising aLCDR1, a LCDR2, and a LCDR3 comprising amino acid sequences at least 90%(such as at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%) identical to amino acids amino acids 27-32, 50-52, and 89-97,respectively, of SEQ ID NO: 12, and can specifically bind to MERS-CoV Sprotein and neutralize MERS-CoV infection. In additional embodiments,the antibody or antigen binding fragment includes a V_(H) comprising aHCDR1, a HCDR2, and a HCDR3 comprising amino acid sequences at least 90%(such as at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%) identical to amino acids 26-33, 51-58, and 97-106,respectively, of SEQ ID NO: 10, and a V_(L) comprising a LCDR1, a LCDR2,and a LCDR3 comprising amino acid sequences at least 90% (such as atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to amino acids amino acids 27-32, 50-52, and 89-97,respectively, of SEQ ID NO: 12, and can specifically bind to MERS-CoV Sprotein and neutralize MERS-CoV infection.

In some embodiments, the antibody or antigen binding fragment includes aV_(H) comprising an amino acid sequence at least 90% (such as at least95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to the amino acid sequence set forth as SEQ ID NO: 10, and canspecifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In more embodiments, the antibody or antigen binding fragmentincludes a V_(L) comprising an amino acid sequence at least 90% (such asat least 95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to the amino acid sequence set forth as SEQ ID NO: 12, and canspecifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In additional embodiments, the antibody or antigen bindingfragment includes a V_(H) and a V_(L) independently comprising aminoacid sequences at least 90% (such as at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%) identical to the amino acidsequences set forth as SEQ ID NOs: 10 and 12, respectively, and canspecifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection.

In additional embodiments, the antibody or antigen binding fragmentincludes a V_(H) comprising the amino acid sequence set forth as one ofSEQ ID NO: 10, and can specifically bind to MERS-CoV S protein andneutralize MERS-CoV infection. In more embodiments, the antibody orantigen binding fragment includes a V_(L) comprising the amino acidsequence set forth as SEQ ID NO: 12, and can specifically bind toMERS-CoV S protein and neutralize MERS-CoV infection. In someembodiments, the antibody or antigen binding fragment includes a V_(H)and a V_(L) comprising the amino acid sequences set forth as SEQ ID NOs:10 and 12, respectively, and can specifically bind to MERS-CoV S proteinand neutralize MERS-CoV infection.

C2

In some embodiments, the antibody or antigen binding fragment can bebased on or derived from the C2 antibody. For example, the antibody orantigen binding fragment can comprise a V_(H) and a V_(L) comprising theHCDR1, the HCDR2, and the HCDR3, and the LCDR1, the LCDR2, and theLCDR3, respectively (for example, according to IMGT), of the C2antibody, and can specifically bind to MERS-CoV S protein and neutralizeMERS-CoV infection.

In some embodiments, the antibody or antigen binding fragment cancomprise a V_(H) comprising the HCDR1, the HCDR2, and the HCDR3 of theC2 V_(H) as set forth in Table 1, and can specifically bind to MERS-CoVS protein and neutralize MERS-CoV infection. In some embodiments, theantibody or antigen binding fragment can comprise a V_(L) comprising theLCDR1, the LCDR2, and the LCDR3 of the C2 V_(L) as set forth in Table 1,and can specifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In some embodiments, the antibody or antigen binding fragmentcan comprise a V_(H) and a V_(L) comprising the HCDR1, the HCDR2, theHCDR3, the LCDR1, the LCDR2, and the LCDR3 of the C2 V_(H) and V_(L) asset forth in Table 1, and can specifically bind to MERS-CoV S proteinand neutralize MERS-CoV infection.

In some embodiments, an antibody or antigen binding fragment based on orderived from the C2 antibody can comprise a V_(L) comprising a glycineto serine substitution or glycine to alanine substitution at kabatposition 29. For example, in some embodiments, the antibody or antigenbinding fragment can comprise a V_(H) comprising the HCDR1, the HCDR2,and the HCDR3 of the C2 V_(H) as set forth in Table 1, and a V_(L)comprising the LCDR1, the LCDR2, and the LCDR3 of the C2 LCDR1 NG-NSV_(L) as set forth in Table 1, and can specifically bind to MERS-CoV Sprotein and neutralize MERS-CoV infection. In some embodiments, theantibody or antigen binding fragment can comprise a V_(H) comprising theHCDR1, the HCDR2, and the HCDR3 of the C2 V_(H) as set forth in Table 1,and a V_(L) comprising the LCDR1, the LCDR2, and the LCDR3 of the C2LCDR1 NG-NA V_(L) as set forth in Table 1, and can specifically bind toMERS-CoV S protein and neutralize MERS-CoV infection.

In some embodiments, the antibody or antigen binding fragment includesat least one CDR (such as an HCDR3) with a sequence that has at least95% (such as at least 96%, at least 97%, at least 98%, at least 99%, oreven 100%) sequence identity to any one of the heavy or light chain CDRsof the C2 V_(H) or V_(L) as shown in Table 1, and can specifically bindto MERS-CoV S protein and neutralize MERS-CoV infection. In someembodiments, the antibody or antigen binding fragment includes a V_(H)comprising a HCDR1, a HCDR2, and a HCDR3 comprising amino acid sequencesat least 90% (such as at least 95%, at least 96%, at least 97%, at least98%, or at least 99%) identical to amino acids 26-33, 51-58, and 97-116,respectively, of SEQ ID NO: 36, and can specifically bind to MERS-CoV Sprotein and neutralize MERS-CoV infection. In some embodiments, theantibody or antigen binding fragment includes a V_(L) comprising aLCDR1, a LCDR2, and a LCDR3 comprising amino acid sequences at least 90%(such as at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%) identical to amino acids amino acids 27-37, 55-57, and94-101, respectively, of SEQ ID NO: 38, and can specifically bind toMERS-CoV S protein and neutralize MERS-CoV infection. In someembodiments, the antibody or antigen binding fragment includes a V_(L)comprising a LCDR1, a LCDR2, and a LCDR3 comprising amino acid sequencesat least 90% (such as at least 95%, at least 96%, at least 97%, at least98%, or at least 99%) identical to amino acids amino acids 27-37, 55-57,and 94-101, respectively, of SEQ ID NO: 110, and can specifically bindto MERS-CoV S protein and neutralize MERS-CoV infection. In someembodiments, the antibody or antigen binding fragment includes a V_(L)comprising a LCDR1, a LCDR2, and a LCDR3 comprising amino acid sequencesat least 90% (such as at least 95%, at least 96%, at least 97%, at least98%, or at least 99%) identical to amino acids amino acids 27-37, 55-57,and 94-101, respectively, of SEQ ID NO: 111, and can specifically bindto MERS-CoV S protein and neutralize MERS-CoV infection. In additionalembodiments, the antibody or antigen binding fragment includes a V_(H)comprising a HCDR1, a HCDR2, and a HCDR3 comprising amino acid sequencesat least 90% (such as at least 95%, at least 96%, at least 97%, at least98%, or at least 99%) identical to amino acids 26-33, 51-58, and 97-116,respectively, of SEQ ID NO: 36, and a V_(L) comprising a LCDR1, a LCDR2,and a LCDR3 comprising amino acid sequences at least 90% (such as atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to amino acids amino acids 27-37, 55-57, and 94-101,respectively, of SEQ ID NO: 38, and can specifically bind to MERS-CoV Sprotein and neutralize MERS-CoV infection. In additional embodiments,the antibody or antigen binding fragment includes a V_(H) comprising aHCDR1, a HCDR2, and a HCDR3 comprising amino acid sequences at least 90%(such as at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%) identical to amino acids 26-33, 51-58, and 97-116,respectively, of SEQ ID NO: 36, and a V_(L) comprising a LCDR1, a LCDR2,and a LCDR3 comprising amino acid sequences at least 90% (such as atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to amino acids amino acids 27-37, 55-57, and 94-101,respectively, of SEQ ID NO: 113, and can specifically bind to MERS-CoV Sprotein and neutralize MERS-CoV infection. In additional embodiments,the antibody or antigen binding fragment includes a V_(H) comprising aHCDR1, a HCDR2, and a HCDR3 comprising amino acid sequences at least 90%(such as at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%) identical to amino acids 26-33, 51-58, and 97-116,respectively, of SEQ ID NO: 36, and a V_(L) comprising a LCDR1, a LCDR2,and a LCDR3 comprising amino acid sequences at least 90% (such as atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to amino acids amino acids 27-37, 55-57, and 94-101,respectively, of SEQ ID NO: 114, and can specifically bind to MERS-CoV Sprotein and neutralize MERS-CoV infection.

In some embodiments, the antibody or antigen binding fragment includes aV_(H) comprising an amino acid sequence at least 90% (such as at least95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to the amino acid sequence set forth as SEQ ID NO: 36, and canspecifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In more embodiments, the antibody or antigen binding fragmentincludes a V_(L) comprising an amino acid sequence at least 90% (such asat least 95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to the amino acid sequence set forth as SEQ ID NO: 38, and canspecifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In more embodiments, the antibody or antigen binding fragmentincludes a V_(L) comprising an amino acid sequence at least 90% (such asat least 95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to the amino acid sequence set forth as SEQ ID NO: 110, andcan specifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In more embodiments, the antibody or antigen binding fragmentincludes a V_(L) comprising an amino acid sequence at least 90% (such asat least 95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to the amino acid sequence set forth as SEQ ID NO: 111, andcan specifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In additional embodiments, the antibody or antigen bindingfragment includes a V_(H) and a V_(L) independently comprising aminoacid sequences at least 90% (such as at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%) identical to the amino acidsequences set forth as SEQ ID NOs: 36 and 38, respectively, and canspecifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In additional embodiments, the antibody or antigen bindingfragment includes a V_(H) and a V_(L) independently comprising aminoacid sequences at least 90% (such as at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%) identical to the amino acidsequences set forth as SEQ ID NO: 36 and 110, respectively, and canspecifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In additional embodiments, the antibody or antigen bindingfragment includes a V_(H) and a V_(L) independently comprising aminoacid sequences at least 90% (such as at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%) identical to the amino acidsequences set forth as SEQ ID NO: 36 and 111, respectively, and canspecifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection.

In additional embodiments, the antibody or antigen binding fragmentincludes a V_(H) comprising the amino acid sequence set forth as one ofSEQ ID NO: 36, and can specifically bind to MERS-CoV S protein andneutralize MERS-CoV infection. In more embodiments, the antibody orantigen binding fragment includes a V_(L) comprising the amino acidsequence set forth as SEQ ID NO: 38, and can specifically bind toMERS-CoV S protein and neutralize MERS-CoV infection. In moreembodiments, the antibody or antigen binding fragment includes a V_(L)comprising the amino acid sequence set forth as SEQ ID NO: 113, and canspecifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In more embodiments, the antibody or antigen binding fragmentincludes a V_(L) comprising the amino acid sequence set forth as SEQ IDNO: 114, and can specifically bind to MERS-CoV S protein and neutralizeMERS-CoV infection. In some embodiments, the antibody or antigen bindingfragment includes a V_(H) and a V_(L) comprising the amino acidsequences set forth as SEQ ID NOs: 36 and 38, respectively, and canspecifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In some embodiments, the antibody or antigen binding fragmentincludes a V_(H) and a V_(L) comprising the amino acid sequences setforth as SEQ ID NOs: 36 and 110, respectively, and can specifically bindto MERS-CoV S protein and neutralize MERS-CoV infection. In someembodiments, the antibody or antigen binding fragment includes a V_(H)and a V_(L) comprising the amino acid sequences set forth as SEQ ID NOs:36 and 111, respectively, and can specifically bind to MERS-CoV Sprotein and neutralize MERS-CoV infection.

C5

In some embodiments, the antibody or antigen binding fragment can bebased on or derived from the C5 antibody. For example, the antibody orantigen binding fragment can comprise a V_(H) and a V_(L) comprising theHCDR1, the HCDR2, and the HCDR3, and the LCDR1, the LCDR2, and theLCDR3, respectively (for example, according to IMGT), of the C5antibody, and can specifically bind to MERS-CoV S protein and neutralizeMERS-CoV infection.

In some embodiments, the antibody or antigen binding fragment cancomprise a V_(H) comprising the HCDR1, the HCDR2, and the HCDR3 of theC5 V_(H) as set forth in Table 1, and can specifically bind to MERS-CoVS protein and neutralize MERS-CoV infection. In some embodiments, theantibody or antigen binding fragment can comprise a V_(L) comprising theLCDR1, the LCDR2, and the LCDR3 of the C5 V_(L) as set forth in Table 1,and can specifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In some embodiments, the antibody or antigen binding fragmentcan comprise a V_(H) and a V_(L) comprising the HCDR1, the HCDR2, theHCDR3, the LCDR1, the LCDR2, and the LCDR3 of the C5 V_(H) and V_(L) asset forth in Table 1, and can specifically bind to MERS-CoV S proteinand neutralize MERS-CoV infection.

In some embodiments, the antibody or antigen binding fragment includesat least one CDR (such as an HCDR3) with a sequence that has at least95% (such as at least 96%, at least 97%, at least 98%, at least 99%, oreven 100%) sequence identity to any one of the heavy or light chain CDRsof the C5 V_(H) or V_(L) as shown in Table 1, and can specifically bindto MERS-CoV S protein and neutralize MERS-CoV infection. In someembodiments, the antibody or antigen binding fragment includes a V_(H)comprising a HCDR1, a HCDR2, and a HCDR3 comprising amino acid sequencesat least 90% (such as at least 95%, at least 96%, at least 97%, at least98%, or at least 99%) identical to amino acids 26-35, 53-59, and 98-115,respectively, of SEQ ID NO: 40, and can specifically bind to MERS-CoV Sprotein and neutralize MERS-CoV infection. In some embodiments, theantibody or antigen binding fragment includes a V_(L) comprising aLCDR1, a LCDR2, and a LCDR3 comprising amino acid sequences at least 90%(such as at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%) identical to amino acids amino acids 26-34, 52-54, and91-100, respectively, of SEQ ID NO: 42, and can specifically bind toMERS-CoV S protein and neutralize MERS-CoV infection. In additionalembodiments, the antibody or antigen binding fragment includes a V_(H)comprising a HCDR1, a HCDR2, and a HCDR3 comprising amino acid sequencesat least 90% (such as at least 95%, at least 96%, at least 97%, at least98%, or at least 99%) identical to amino acids 26-35, 53-59, and 98-115,respectively, of SEQ ID NO: 40, and a V_(L) comprising a LCDR1, a LCDR2,and a LCDR3 comprising amino acid sequences at least 90% (such as atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to amino acids amino acids 26-34, 52-54, and 91-100,respectively, of SEQ ID NO: 42, and can specifically bind to MERS-CoV Sprotein and neutralize MERS-CoV infection.

In some embodiments, the antibody or antigen binding fragment includes aV_(H) comprising an amino acid sequence at least 90% (such as at least95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to the amino acid sequence set forth as SEQ ID NO: 40, and canspecifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In more embodiments, the antibody or antigen binding fragmentincludes a V_(L) comprising an amino acid sequence at least 90% (such asat least 95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to the amino acid sequence set forth as SEQ ID NO: 42, and canspecifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In additional embodiments, the antibody or antigen bindingfragment includes a V_(H) and a V_(L) independently comprising aminoacid sequences at least 90% (such as at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%) identical to the amino acidsequences set forth as SEQ ID NOs: 40 and 42, respectively, and canspecifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection.

In additional embodiments, the antibody or antigen binding fragmentincludes a V_(H) comprising the amino acid sequence set forth as one ofSEQ ID NO: 40, and can specifically bind to MERS-CoV S protein andneutralize MERS-CoV infection. In more embodiments, the antibody orantigen binding fragment includes a V_(L) comprising the amino acidsequence set forth as SEQ ID NO: 42, and can specifically bind toMERS-CoV S protein and neutralize MERS-CoV infection. In someembodiments, the antibody or antigen binding fragment includes a V_(H)and a V_(L) comprising the amino acid sequences set forth as SEQ ID NOs:40 and 42, respectively, and can specifically bind to MERS-CoV S proteinand neutralize MERS-CoV infection.

A2

In some embodiments, the antibody or antigen binding fragment can bebased on or derived from the A2 antibody. For example, the antibody orantigen binding fragment can comprise a V_(H) and a V_(L) comprising theHCDR1, the HCDR2, and the HCDR3, and the LCDR1, the LCDR2, and theLCDR3, respectively (for example, according to IMGT), of the A2antibody, and can specifically bind to MERS-CoV S protein and neutralizeMERS-CoV infection.

In some embodiments, the antibody or antigen binding fragment cancomprise a V_(H) comprising the HCDR1, the HCDR2, and the HCDR3 of theA2 V_(H) as set forth in Table 1, and can specifically bind to MERS-CoVS protein and neutralize MERS-CoV infection. In some embodiments, theantibody or antigen binding fragment can comprise a V_(L) comprising theLCDR1, the LCDR2, and the LCDR3 of the A2 V_(L) as set forth in Table 1,and can specifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In some embodiments, the antibody or antigen binding fragmentcan comprise a V_(H) and a V_(L) comprising the HCDR1, the HCDR2, theHCDR3, the LCDR1, the LCDR2, and the LCDR3 of the A2 V_(H) and V_(L) asset forth in Table 1, and can specifically bind to MERS-CoV S proteinand neutralize MERS-CoV infection. In some embodiments, the antibody orantigen binding fragment includes at least one CDR (such as an HCDR3)with a sequence that has at least 95% (such as at least 96%, at least97%, at least 98%, at least 99%, or even 100%) sequence identity to anyone of the heavy or light chain CDRs of the A2 V_(H) or V_(L) as shownin Table 1, and can specifically bind to MERS-CoV S protein andneutralize MERS-CoV infection. In some embodiments, the antibody orantigen binding fragment includes a V_(H) comprising a HCDR1, a HCDR2,and a HCDR3 comprising amino acid sequences at least 90% (such as atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to amino acids 26-33, 51-58, and 97-111, respectively, of SEQID NO: 44, and can specifically bind to MERS-CoV S protein andneutralize MERS-CoV infection. In some embodiments, the antibody orantigen binding fragment includes a V_(L) comprising a LCDR1, a LCDR2,and a LCDR3 comprising amino acid sequences at least 90% (such as atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to amino acids amino acids 26-34, 52-54, and 91-101,respectively, of SEQ ID NO: 46, and can specifically bind to MERS-CoV Sprotein and neutralize MERS-CoV infection. In additional embodiments,the antibody or antigen binding fragment includes a V_(H) comprising aHCDR1, a HCDR2, and a HCDR3 comprising amino acid sequences at least 90%(such as at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%) identical to amino acids 26-33, 51-58, and 97-111,respectively, of SEQ ID NO: 44, and a V_(L) comprising a LCDR1, a LCDR2,and a LCDR3 comprising amino acid sequences at least 90% (such as atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to amino acids amino acids 26-34, 52-54, and 91-101,respectively, of SEQ ID NO: 46, and can specifically bind to MERS-CoV Sprotein and neutralize MERS-CoV infection.

In some embodiments, the antibody or antigen binding fragment includes aV_(H) comprising an amino acid sequence at least 90% (such as at least95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to the amino acid sequence set forth as SEQ ID NO: 44, and canspecifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In more embodiments, the antibody or antigen binding fragmentincludes a V_(L) comprising an amino acid sequence at least 90% (such asat least 95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to the amino acid sequence set forth as SEQ ID NO: 46, and canspecifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In additional embodiments, the antibody or antigen bindingfragment includes a V_(H) and a V_(L) independently comprising aminoacid sequences at least 90% (such as at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%) identical to the amino acidsequences set forth as SEQ ID NO: 44 and 46, respectively, and canspecifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection.

In additional embodiments, the antibody or antigen binding fragmentincludes a V_(H) comprising the amino acid sequence set forth as one ofSEQ ID NO: 44, and can specifically bind to MERS-CoV S protein andneutralize MERS-CoV infection. In more embodiments, the antibody orantigen binding fragment includes a V_(L) comprising the amino acidsequence set forth as SEQ ID NO: 46, and can specifically bind toMERS-CoV S protein and neutralize MERS-CoV infection. In someembodiments, the antibody or antigen binding fragment includes a V_(H)and a V_(L) comprising the amino acid sequences set forth as SEQ ID NOs:44 and 46, respectively, and can specifically bind to MERS-CoV S proteinand neutralize MERS-CoV infection.

A10

In some embodiments, the antibody or antigen binding fragment can bebased on or derived from the A10 antibody. For example, the antibody orantigen binding fragment can comprise a V_(H) and a V_(L) comprising theHCDR1, the HCDR2, and the HCDR3, and the LCDR1, the LCDR2, and theLCDR3, respectively (for example, according to IMGT), of the A10antibody, and can specifically bind to MERS-CoV S protein and neutralizeMERS-CoV infection.

In some embodiments, the antibody or antigen binding fragment cancomprise a V_(H) comprising the HCDR1, the HCDR2, and the HCDR3 of theA10 V_(H) as set forth in Table 1, and can specifically bind to MERS-CoVS protein and neutralize MERS-CoV infection. In some embodiments, theantibody or antigen binding fragment can comprise a V_(L) comprising theLCDR1, the LCDR2, and the LCDR3 of the A10 V_(L) as set forth in Table1, and can specifically bind to MERS-CoV S protein and neutralizeMERS-CoV infection. In some embodiments, the antibody or antigen bindingfragment can comprise a V_(H) and a V_(L) comprising the HCDR1, theHCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 of the A10 V_(H)and V_(L) as set forth in Table 1, and can specifically bind to MERS-CoVS protein and neutralize MERS-CoV infection.

In some embodiments, the antibody or antigen binding fragment includesat least one CDR (such as an HCDR3) with a sequence that has at least95% (such as at least 96%, at least 97%, at least 98%, at least 99%, oreven 100%) sequence identity to any one of the heavy or light chain CDRsof the A10 V_(H) or V_(L) as shown in Table 1, and can specifically bindto MERS-CoV S protein and neutralize MERS-CoV infection. In someembodiments, the antibody or antigen binding fragment includes a V_(H)comprising a HCDR1, a HCDR2, and a HCDR3 comprising amino acid sequencesat least 90% (such as at least 95%, at least 96%, at least 97%, at least98%, or at least 99%) identical to amino acids 26-33, 51-58, and 97-111,respectively, of SEQ ID NO: 48, and can specifically bind to MERS-CoV Sprotein and neutralize MERS-CoV infection. In some embodiments, theantibody or antigen binding fragment includes a V_(L) comprising aLCDR1, a LCDR2, and a LCDR3 comprising amino acid sequences at least 90%(such as at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%) identical to amino acids amino acids 26-34, 52-54, and92-102, respectively, of SEQ ID NO: 50, and can specifically bind toMERS-CoV S protein and neutralize MERS-CoV infection. In additionalembodiments, the antibody or antigen binding fragment includes a V_(H)comprising a HCDR1, a HCDR2, and a HCDR3 comprising amino acid sequencesat least 90% (such as at least 95%, at least 96%, at least 97%, at least98%, or at least 99%) identical to amino acids 26-33, 51-58, and 97-111,respectively, of SEQ ID NO: 48, and a V_(L) comprising a LCDR1, a LCDR2,and a LCDR3 comprising amino acid sequences at least 90% (such as atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to amino acids amino acids 26-34, 52-54, and 92-102,respectively, of SEQ ID NO: 50, and can specifically bind to MERS-CoV Sprotein and neutralize MERS-CoV infection.

In some embodiments, the antibody or antigen binding fragment includes aV_(H) comprising an amino acid sequence at least 90% (such as at least95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to the amino acid sequence set forth as SEQ ID NO: 48, and canspecifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In more embodiments, the antibody or antigen binding fragmentincludes a V_(L) comprising an amino acid sequence at least 90% (such asat least 95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to the amino acid sequence set forth as SEQ ID NO: 50, and canspecifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In additional embodiments, the antibody or antigen bindingfragment includes a V_(H) and a V_(L) independently comprising aminoacid sequences at least 90% (such as at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%) identical to the amino acidsequences set forth as SEQ ID NOs: 48 and 50, respectively, and canspecifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection.

In additional embodiments, the antibody or antigen binding fragmentincludes a V_(H) comprising the amino acid sequence set forth as one ofSEQ ID NO: 48, and can specifically bind to MERS-CoV S protein andneutralize MERS-CoV infection. In more embodiments, the antibody orantigen binding fragment includes a V_(L) comprising the amino acidsequence set forth as SEQ ID NO: 50, and can specifically bind toMERS-CoV S protein and neutralize MERS-CoV infection. In someembodiments, the antibody or antigen binding fragment includes a V_(H)and a V_(L) comprising the amino acid sequences set forth as SEQ ID NOs:48 and 50, respectively, and can specifically bind to MERS-CoV S proteinand neutralize MERS-CoV infection.

FIB_B2

In some embodiments, the antibody or antigen binding fragment can bebased on or derived from the FIB_B2 antibody. For example, the antibodyor antigen binding fragment can comprise a V_(H) and a V_(L) comprisingthe HCDR1, the HCDR2, and the HCDR3, and the LCDR1, the LCDR2, and theLCDR3, respectively (for example, according to IMGT), of the FIB_B2antibody, and can specifically bind to MERS-CoV S protein and neutralizeMERS-CoV infection.

In some embodiments, the antibody or antigen binding fragment cancomprise a V_(H) comprising the HCDR1, the HCDR2, and the HCDR3 of theFIB_B2 V_(H) as set forth in Table 1, and can specifically bind toMERS-CoV S protein and neutralize MERS-CoV infection. In someembodiments, the antibody or antigen binding fragment can comprise aV_(L) comprising the LCDR1, the LCDR2, and the LCDR3 of the FIB_B2 V_(L)as set forth in Table 1, and can specifically bind to MERS-CoV S proteinand neutralize MERS-CoV infection. In some embodiments, the antibody orantigen binding fragment can comprise a V_(H) and a V_(L) comprising theHCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 of theFIB_B2 V_(H) and V_(L) as set forth in Table 1, and can specificallybind to MERS-CoV S protein and neutralize MERS-CoV infection.

In some embodiments, the antibody or antigen binding fragment includesat least one CDR (such as an HCDR3) with a sequence that has at least95% (such as at least 96%, at least 97%, at least 98%, at least 99%, oreven 100%) sequence identity to any one of the heavy or light chain CDRsof the FIB_B2 V_(H) or V_(L) as shown in Table 1, and can specificallybind to MERS-CoV S protein and neutralize MERS-CoV infection. In someembodiments, the antibody or antigen binding fragment includes a V_(H)comprising a HCDR1, a HCDR2, and a HCDR3 comprising amino acid sequencesat least 90% (such as at least 95%, at least 96%, at least 97%, at least98%, or at least 99%) identical to amino acids 26-33, 51-55, and 97-107,respectively, of SEQ ID NO: 52, and can specifically bind to MERS-CoV Sprotein and neutralize MERS-CoV infection. In some embodiments, theantibody or antigen binding fragment includes a V_(L) comprising aLCDR1, a LCDR2, and a LCDR3 comprising amino acid sequences at least 90%(such as at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%) identical to amino acids amino acids 27-32, 50-52, and 89-97,respectively, of SEQ ID NO: 54, and can specifically bind to MERS-CoV Sprotein and neutralize MERS-CoV infection. In additional embodiments,the antibody or antigen binding fragment includes a V_(H) comprising aHCDR1, a HCDR2, and a HCDR3 comprising amino acid sequences at least 90%(such as at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%) identical to amino acids 26-33, 51-55, and 97-107,respectively, of SEQ ID NO: 52, and a V_(L) comprising a LCDR1, a LCDR2,and a LCDR3 comprising amino acid sequences at least 90% (such as atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to amino acids amino acids 27-32, 50-52, and 89-97,respectively, of SEQ ID NO: 54, and can specifically bind to MERS-CoV Sprotein and neutralize MERS-CoV infection.

In some embodiments, the antibody or antigen binding fragment includes aV_(H) comprising an amino acid sequence at least 90% (such as at least95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to the amino acid sequence set forth as SEQ ID NO: 52, and canspecifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In more embodiments, the antibody or antigen binding fragmentincludes a V_(L) comprising an amino acid sequence at least 90% (such asat least 95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to the amino acid sequence set forth as SEQ ID NO: 54, and canspecifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In additional embodiments, the antibody or antigen bindingfragment includes a V_(H) and a V_(L) independently comprising aminoacid sequences at least 90% (such as at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%) identical to the amino acidsequences set forth as SEQ ID NO: 52 and 54, respectively, and canspecifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection.

In additional embodiments, the antibody or antigen binding fragmentincludes a V_(H) comprising the amino acid sequence set forth as one ofSEQ ID NO: 52, and can specifically bind to MERS-CoV S protein andneutralize MERS-CoV infection. In more embodiments, the antibody orantigen binding fragment includes a V_(L) comprising the amino acidsequence set forth as SEQ ID NO: 54, and can specifically bind toMERS-CoV S protein and neutralize MERS-CoV infection. In someembodiments, the antibody or antigen binding fragment includes a V_(H)and a V_(L) comprising the amino acid sequences set forth as SEQ ID NOs:52 and 54, respectively, and can specifically bind to MERS-CoV S proteinand neutralize MERS-CoV infection.

FIB_H1

In some embodiments, the antibody or antigen binding fragment can bebased on or derived from the FIB_H1 antibody. For example, the antibodyor antigen binding fragment can comprise a V_(H) and a V_(L) comprisingthe HCDR1, the HCDR2, and the HCDR3, and the LCDR1, the LCDR2, and theLCDR3, respectively (for example, according to IMGT), of the FIB_H1antibody, and can specifically bind to MERS-CoV S protein and neutralizeMERS-CoV infection.

In some embodiments, the antibody or antigen binding fragment cancomprise a V_(H) comprising the HCDR1, the HCDR2, and the HCDR3, of theFIB_H1 V_(H) as set forth in Table 1, and can specifically bind toMERS-CoV S protein and neutralize MERS-CoV infection. In someembodiments, the antibody or antigen binding fragment can comprise aV_(L) comprising the LCDR1, the LCDR2, and the LCDR3 of the FIB_H1 V_(L)as set forth in Table 1, and can specifically bind to MERS-CoV S proteinand neutralize MERS-CoV infection. In some embodiments, the antibody orantigen binding fragment can comprise a V_(H) and a V_(L) comprising theHCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 of theFIB_H1 V_(H) and V_(L) as set forth in Table 1, and can specificallybind to MERS-CoV S protein and neutralize MERS-CoV infection.

In some embodiments, the antibody or antigen binding fragment includesat least one CDR (such as an HCDR3) with a sequence that has at least95% (such as at least 96%, at least 97%, at least 98%, at least 99%, oreven 100%) sequence identity to any one of the heavy or light chain CDRsof the FIB_H1 V_(H) or V_(L) as shown in Table 1, and can specificallybind to MERS-CoV S protein and neutralize MERS-CoV infection. In someembodiments, the antibody or antigen binding fragment includes a V_(H)comprising a HCDR1, a HCDR2, and a HCDR3 comprising amino acid sequencesat least 90% (such as at least 95%, at least 96%, at least 97%, at least98%, or at least 99%) identical to amino acids 26-33, 51-55, and 97-107,respectively, of SEQ ID NO: 56, and can specifically bind to MERS-CoV Sprotein and neutralize MERS-CoV infection. In some embodiments, theantibody or antigen binding fragment includes a V_(L) comprising aLCDR1, a LCDR2, and a LCDR3 comprising amino acid sequences at least 90%(such as at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%) identical to amino acids amino acids 27-32, 50-52, and 89-97,respectively, of SEQ ID NO: 58, and can specifically bind to MERS-CoV Sprotein and neutralize MERS-CoV infection. In additional embodiments,the antibody or antigen binding fragment includes a V_(H) comprising aHCDR1, a HCDR2, and a HCDR3 comprising amino acid sequences at least 90%(such as at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%) identical to amino acids 26-33, 51-55, and 97-107,respectively, of SEQ ID NO: 56, and a V_(L) comprising a LCDR1, a LCDR2,and a LCDR3 comprising amino acid sequences at least 90% (such as atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to amino acids amino acids 27-32, 50-52, and 89-97,respectively, of SEQ ID NO: 58, and can specifically bind to MERS-CoV Sprotein and neutralize MERS-CoV infection.

In some embodiments, the antibody or antigen binding fragment includes aV_(H) comprising an amino acid sequence at least 90% (such as at least95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to the amino acid sequence set forth as SEQ ID NO: 56, and canspecifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In more embodiments, the antibody or antigen binding fragmentincludes a V_(L) comprising an amino acid sequence at least 90% (such asat least 95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to the amino acid sequence set forth as SEQ ID NO: 58, and canspecifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In additional embodiments, the antibody or antigen bindingfragment includes a V_(H) and a V_(L) independently comprising aminoacid sequences at least 90% (such as at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%) identical to the amino acidsequences set forth as SEQ ID NO: 56 and 58, respectively.

In additional embodiments, the antibody or antigen binding fragmentincludes a V_(H) comprising the amino acid sequence set forth as one ofSEQ ID NO: 56, and can specifically bind to MERS-CoV S protein andneutralize MERS-CoV infection. In more embodiments, the antibody orantigen binding fragment includes a V_(L) comprising the amino acidsequence set forth as SEQ ID NO: 58, and can specifically bind toMERS-CoV S protein and neutralize MERS-CoV infection. In someembodiments, the antibody or antigen binding fragment includes a V_(H)and a V_(L) comprising the amino acid sequences set forth as SEQ ID NOs:56 and 58, respectively, and can specifically bind to MERS-CoV S proteinand neutralize MERS-CoV infection.

G2

In some embodiments, the antibody or antigen binding fragment can bebased on or derived from the G2 antibody. For example, the antibody orantigen binding fragment can comprise a V_(H) and a V_(L) comprising theHCDR1, the HCDR2, and the HCDR3, and the LCDR1, the LCDR2, and theLCDR3, respectively (for example, according to IMGT), of the G2antibody, and can specifically bind to MERS-CoV S protein and neutralizeMERS-CoV infection.

In some embodiments, the antibody or antigen binding fragment cancomprise a V_(H) comprising the HCDR1, the HCDR2, and the HCDR3, of theG2 V_(H) as set forth in Table 1, and can specifically bind to MERS-CoVS protein and neutralize MERS-CoV infection. In some embodiments, theantibody or antigen binding fragment can comprise a V_(L) comprising theLCDR1, the LCDR2, and the LCDR3 of the G2 V_(L) as set forth in Table 1,and can specifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In some embodiments, the antibody or antigen binding fragmentcan comprise a V_(H) and a V_(L) comprising the HCDR1, the HCDR2, theHCDR3, the LCDR1, the LCDR2, and the LCDR3 of the G2 V_(H) and V_(L) asset forth in Table 1, and can specifically bind to MERS-CoV S proteinand neutralize MERS-CoV infection.

In some embodiments, the antibody or antigen binding fragment includesat least one CDR (such as an HCDR3) with a sequence that has at least90% (such as at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or even 100%) sequence identity to any one of the heavy orlight chain CDRs of the G2 V_(H) or V_(L) as shown in Table 1, and canspecifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In some embodiments, the antibody or antigen binding fragmentincludes a V_(H) comprising a HCDR1, a HCDR2, and a HCDR3 comprisingamino acid sequences at least 90% (such as at least 95%, at least 96%,at least 97%, at least 98%, at least 99%, or even 100%) identical toamino acids 26-33, 51-58, and 97-107, respectively, of SEQ ID NO: 115,and can specifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In some embodiments, the antibody or antigen binding fragmentincludes a V_(L) comprising a LCDR1, a LCDR2, and a LCDR3 comprisingamino acid sequences at least 90% (such as at least 95%, at least 96%,at least 97%, at least 98%, at least 99%, or even 100%) identical toamino acids amino acids 26-36, 54-56, and 93-97, respectively, of SEQ IDNO: 117, and can specifically bind to MERS-CoV S protein and neutralizeMERS-CoV infection. In additional embodiments, the antibody or antigenbinding fragment includes a V_(H) comprising a HCDR1, a HCDR2, and aHCDR3 comprising amino acid sequences at least 90% (such as at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or even100%) identical to amino acids 26-33, 51-58, and 97-107, respectively,of SEQ ID NO: 115, and a V_(L) comprising a LCDR1, a LCDR2, and a LCDR3comprising amino acid sequences at least 90% (such as at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or even 100%)identical to amino acids amino acids 26-36, 54-56, and 93-97,respectively, of SEQ ID NO: 117, and can specifically bind to MERS-CoV Sprotein and neutralize MERS-CoV infection.

In some embodiments, the antibody or antigen binding fragment includes aV_(H) comprising an amino acid sequence at least 90% (such as at least95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to the amino acid sequence set forth as SEQ ID NO: 115, andcan specifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In more embodiments, the antibody or antigen binding fragmentincludes a V_(L) comprising an amino acid sequence at least 90% (such asat least 95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to the amino acid sequence set forth as SEQ ID NO: 117, andcan specifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In additional embodiments, the antibody or antigen bindingfragment includes a V_(H) and a V_(L) independently comprising aminoacid sequences at least 90% (such as at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%) identical to the amino acidsequences set forth as SEQ ID NO: 115 and 117, respectively.

In additional embodiments, the antibody or antigen binding fragmentincludes a V_(H) comprising the amino acid sequence set forth as one ofSEQ ID NO: 115, and can specifically bind to MERS-CoV S protein andneutralize MERS-CoV infection. In more embodiments, the antibody orantigen binding fragment includes a V_(L) comprising the amino acidsequence set forth as SEQ ID NO: 117, and can specifically bind toMERS-CoV S protein and neutralize MERS-CoV infection. In someembodiments, the antibody or antigen binding fragment includes a V_(H)and a V_(L) comprising the amino acid sequences set forth as SEQ ID NOs:115 and 117, respectively, and can specifically bind to MERS-CoV Sprotein and neutralize MERS-CoV infection. In some embodiments, a humanchimeric antibody including the V_(H) of the murine G2 antibody and ahuman IgG₁ constant domain can generated by linking the G2 V_(H) (SEQ IDNO: 115) to the human IgG₁ constant domain. The DNA and proteinsequences of the chimeric heavy chain are provided as SEQ ID NOs: 153and 154. The chimeric V_(H) includes a KG-TP substitution at thebeginning of the constant domain to enhance compatibility of the humanheavy chain and the mouse light chain. The G2 mouse-human chimeric mAb(G2-huIgG KG/TP) neutralizes 11 MERS-CoV strains with comparable IC50,but slightly lower IC80 and IC90 to G2, as assayed using a pseudovirusneutralization assay (see the Table 5).

G4

In some embodiments, the antibody or antigen binding fragment can bebased on or derived from the G4 antibody. For example, the antibody orantigen binding fragment can comprise a V_(H) and a V_(L) comprising theHCDR1, the HCDR2, and the HCDR3, and the LCDR1, the LCDR2, and theLCDR3, respectively (for example, according to IMGT), of the G4antibody, and can specifically bind to MERS-CoV S protein and neutralizeMERS-CoV infection.

In some embodiments, the antibody or antigen binding fragment cancomprise a V_(H) comprising the HCDR1, the HCDR2, and the HCDR3 of theG4 V_(H) as set forth in Table 1, and can specifically bind to MERS-CoVS protein and neutralize MERS-CoV infection. In some embodiments, theantibody or antigen binding fragment can comprise a V_(L) comprising theLCDR1, the LCDR2, and the LCDR3 of the G4 V_(L) as set forth in Table 1,and can specifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In some embodiments, the antibody or antigen binding fragmentcan comprise a V_(H) and a V_(L) comprising the HCDR1, the HCDR2, theHCDR3, the LCDR1, the LCDR2, and the LCDR3 of the G4 V_(H) and V_(L) asset forth in Table 1, and can specifically bind to MERS-CoV S proteinand neutralize MERS-CoV infection.

In some embodiments, the antibody or antigen binding fragment includesat least one CDR (such as an HCDR3) with a sequence that has at least90% (such as at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or even 100%) sequence identity to any one of the heavy orlight chain CDRs of the G4 V_(H) or V_(L) as shown in Table 1, and canspecifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In some embodiments, the antibody or antigen binding fragmentincludes a V_(H) comprising a HCDR1, a HCDR2, and a HCDR3 comprisingamino acid sequences at least 90% (such as at least 95%, at least 96%,at least 97%, at least 98%, at least 99%, or even 100%) identical toamino acids 26-33, 51-58, and 97-111, respectively, of SEQ ID NO: 119,and can specifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In some embodiments, the antibody or antigen binding fragmentincludes a V_(L) comprising a LCDR1, a LCDR2, and a LCDR3 comprisingamino acid sequences at least 90% (such as at least 95%, at least 96%,at least 97%, at least 98%, at least 99%, or even 100%) identical toamino acids amino acids 26-36, 54-56, and 93-101, respectively, of SEQID NO: 121, and can specifically bind to MERS-CoV S protein andneutralize MERS-CoV infection. In additional embodiments, the antibodyor antigen binding fragment includes a V_(H) comprising a HCDR1, aHCDR2, and a HCDR3 comprising amino acid sequences at least 90% (such asat least 95%, at least 96%, at least 97%, at least 98%, at least 99%, oreven 100%) identical to amino acids 26-33, 51-58, and 97-111,respectively, of SEQ ID NO: 119, and a V_(L) comprising a LCDR1, aLCDR2, and a LCDR3 comprising amino acid sequences at least 90% (such asat least 95%, at least 96%, at least 97%, at least 98%, at least 99%, oreven 100%) identical to amino acids amino acids 26-36, 54-56, and93-101, respectively, of SEQ ID NO: 121, and can specifically bind toMERS-CoV S protein and neutralize MERS-CoV infection.

In some embodiments, the antibody or antigen binding fragment includes aV_(H) comprising an amino acid sequence at least 90% (such as at least95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to the amino acid sequence set forth as SEQ ID NO: 119, andcan specifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In more embodiments, the antibody or antigen binding fragmentincludes a V_(L) comprising an amino acid sequence at least 90% (such asat least 95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to the amino acid sequence set forth as SEQ ID NO: 121, andcan specifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In additional embodiments, the antibody or antigen bindingfragment includes a V_(H) and a V_(L) independently comprising aminoacid sequences at least 90% (such as at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%) identical to the amino acidsequences set forth as SEQ ID NO: 119 and 121, respectively.

In additional embodiments, the antibody or antigen binding fragmentincludes a V_(H) comprising the amino acid sequence set forth as one ofSEQ ID NO: 119, and can specifically bind to MERS-CoV S protein andneutralize MERS-CoV infection. In more embodiments, the antibody orantigen binding fragment includes a V_(L) comprising the amino acidsequence set forth as SEQ ID NO: 121, and can specifically bind toMERS-CoV S protein and neutralize MERS-CoV infection. In someembodiments, the antibody or antigen binding fragment includes a V_(H)and a V_(L) comprising the amino acid sequences set forth as SEQ ID NOs:119 and 121, respectively, and can specifically bind to MERS-CoV Sprotein and neutralize MERS-CoV infection.

D12

In some embodiments, the antibody or antigen binding fragment can bebased on or derived from the D12 antibody. For example, the antibody orantigen binding fragment can comprise a V_(H) and a V_(L) comprising theHCDR1, the HCDR2, and the HCDR3, and the LCDR1, the LCDR2, and theLCDR3, respectively (for example, according to IMGT), of the D12antibody, and can specifically bind to MERS-CoV S protein and neutralizeMERS-CoV infection.

In some embodiments, the antibody or antigen binding fragment cancomprise a V_(H) comprising the HCDR1, the HCDR2, and the HCDR3, of theD12 V_(H) as set forth in Table 1, and can specifically bind to MERS-CoVS protein and neutralize MERS-CoV infection. In some embodiments, theantibody or antigen binding fragment can comprise a V_(L) comprising theLCDR1, the LCDR2, and the LCDR3 of the D12 V_(L) as set forth in Table1, and can specifically bind to MERS-CoV S protein and neutralizeMERS-CoV infection. In some embodiments, the antibody or antigen bindingfragment can comprise a V_(H) and a V_(L) comprising the HCDR1, theHCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 of the D12 V_(H)and V_(L) as set forth in Table 1, and can specifically bind to MERS-CoVS protein and neutralize MERS-CoV infection.

In some embodiments, the antibody or antigen binding fragment includesat least one CDR (such as an HCDR3) with a sequence that has at least90% (such as at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or even 100%) sequence identity to any one of the heavy orlight chain CDRs of the D12 V_(H) or V_(L) as shown in Table 1, and canspecifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In some embodiments, the antibody or antigen binding fragmentincludes a V_(H) comprising a HCDR1, a HCDR2, and a HCDR3 comprisingamino acid sequences at least 90% (such as at least 95%, at least 96%,at least 97%, at least 98%, at least 99%, or even 100%) identical toamino acids 26-33, 51-58, and 97-105, respectively, of SEQ ID NO: 123,and can specifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In some embodiments, the antibody or antigen binding fragmentincludes a V_(L) comprising a LCDR1, a LCDR2, and a LCDR3 comprisingamino acid sequences at least 90% (such as at least 95%, at least 96%,at least 97%, at least 98%, at least 99%, or even 100%) identical toamino acids amino acids 26-32, 50-52, and 89-97, respectively, of SEQ IDNO: 125, and can specifically bind to MERS-CoV S protein and neutralizeMERS-CoV infection. In additional embodiments, the antibody or antigenbinding fragment includes a V_(H) comprising a HCDR1, a HCDR2, and aHCDR3 comprising amino acid sequences at least 90% (such as at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or even100%) identical to amino acids 26-33, 51-58, and 97-105, respectively,of SEQ ID NO: 123, and a V_(L) comprising a LCDR1, a LCDR2, and a LCDR3comprising amino acid sequences at least 90% (such as at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or even 100%)identical to amino acids amino acids 26-32, 50-52, and 89-97,respectively, of SEQ ID NO: 125, and can specifically bind to MERS-CoV Sprotein and neutralize MERS-CoV infection.

In some embodiments, the antibody or antigen binding fragment includes aV_(H) comprising an amino acid sequence at least 90% (such as at least95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to the amino acid sequence set forth as SEQ ID NO: 123, andcan specifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In more embodiments, the antibody or antigen binding fragmentincludes a V_(L) comprising an amino acid sequence at least 90% (such asat least 95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to the amino acid sequence set forth as SEQ ID NO: 125, andcan specifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In additional embodiments, the antibody or antigen bindingfragment includes a V_(H) and a V_(L) independently comprising aminoacid sequences at least 90% (such as at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%) identical to the amino acidsequences set forth as SEQ ID NO: 123 and 125, respectively.

In additional embodiments, the antibody or antigen binding fragmentincludes a V_(H) comprising the amino acid sequence set forth as one ofSEQ ID NO: 123, and can specifically bind to MERS-CoV S protein andneutralize MERS-CoV infection. In more embodiments, the antibody orantigen binding fragment includes a V_(L) comprising the amino acidsequence set forth as SEQ ID NO: 125, and can specifically bind toMERS-CoV S protein and neutralize MERS-CoV infection. In someembodiments, the antibody or antigen binding fragment includes a V_(H)and a V_(L) comprising the amino acid sequences set forth as SEQ ID NOs:123 and 125, respectively, and can specifically bind to MERS-CoV Sprotein and neutralize MERS-CoV infection.

F11

In some embodiments, the antibody or antigen binding fragment can bebased on or derived from the F11 antibody. For example, the antibody orantigen binding fragment can comprise a V_(H) and a V_(L) comprising theHCDR1, the HCDR2, and the HCDR3, and the LCDR1, the LCDR2, and theLCDR3, respectively (for example, according to IMGT), of the F11antibody, and can specifically bind to MERS-CoV S protein and neutralizeMERS-CoV infection.

In some embodiments, the antibody or antigen binding fragment cancomprise a V_(H) comprising the HCDR1, the HCDR2, and the HCDR3, of theF11 V_(H) as set forth in Table 1, and can specifically bind to MERS-CoVS protein and neutralize MERS-CoV infection. In some embodiments, theantibody or antigen binding fragment can comprise a V_(L) comprising theLCDR1, the LCDR2, and the LCDR3 of the F11 V_(L) as set forth in Table1, and can specifically bind to MERS-CoV S protein and neutralizeMERS-CoV infection. In some embodiments, the antibody or antigen bindingfragment can comprise a V_(H) and a V_(L) comprising the HCDR1, theHCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 of the F11 V_(H)and V_(L) as set forth in Table 1, and can specifically bind to MERS-CoVS protein and neutralize MERS-CoV infection.

In some embodiments, the antibody or antigen binding fragment includesat least one CDR (such as an HCDR3) with a sequence that has at least90% (such as at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or even 100%) sequence identity to any one of the heavy orlight chain CDRs of the F11 V_(H) or V_(L) as shown in Table 1, and canspecifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In some embodiments, the antibody or antigen binding fragmentincludes a V_(H) comprising a HCDR1, a HCDR2, and a HCDR3 comprisingamino acid sequences at least 90% (such as at least 95%, at least 96%,at least 97%, at least 98%, at least 99%, or even 100%) identical toamino acids 26-33, 51-58, and 97-111, respectively, of SEQ ID NO: 127,and can specifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In some embodiments, the antibody or antigen binding fragmentincludes a V_(L) comprising a LCDR1, a LCDR2, and a LCDR3 comprisingamino acid sequences at least 90% (such as at least 95%, at least 96%,at least 97%, at least 98%, at least 99%, or even 100%) identical toamino acids amino acids 26-37, 55-57, and 94-102, respectively, of SEQID NO: 129, and can specifically bind to MERS-CoV S protein andneutralize MERS-CoV infection. In additional embodiments, the antibodyor antigen binding fragment includes a V_(H) comprising a HCDR1, aHCDR2, and a HCDR3 comprising amino acid sequences at least 90% (such asat least 95%, at least 96%, at least 97%, at least 98%, at least 99%, oreven 100%) identical to amino acids 26-33, 51-58, and 97-111,respectively, of SEQ ID NO: 127, and a V_(L) comprising a LCDR1, aLCDR2, and a LCDR3 comprising amino acid sequences at least 90% (such asat least 95%, at least 96%, at least 97%, at least 98%, at least 99%, oreven 100%) identical to amino acids amino acids 26-37, 55-57, and94-102, respectively, of SEQ ID NO: 129, and can specifically bind toMERS-CoV S protein and neutralize MERS-CoV infection.

In some embodiments, the antibody or antigen binding fragment includes aV_(H) comprising an amino acid sequence at least 90% (such as at least95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to the amino acid sequence set forth as SEQ ID NO: 127, andcan specifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In more embodiments, the antibody or antigen binding fragmentincludes a V_(L) comprising an amino acid sequence at least 90% (such asat least 95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to the amino acid sequence set forth as SEQ ID NO: 129, andcan specifically bind to MERS-CoV S protein and neutralize MERS-CoVinfection. In additional embodiments, the antibody or antigen bindingfragment includes a V_(H) and a V_(L) independently comprising aminoacid sequences at least 90% (such as at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%) identical to the amino acidsequences set forth as SEQ ID NO: 127 and 129, respectively.

In additional embodiments, the antibody or antigen binding fragmentincludes a V_(H) comprising the amino acid sequence set forth as one ofSEQ ID NO: 127, and can specifically bind to MERS-CoV S protein andneutralize MERS-CoV infection. In more embodiments, the antibody orantigen binding fragment includes a V_(L) comprising the amino acidsequence set forth as SEQ ID NO: 129, and can specifically bind toMERS-CoV S protein and neutralize MERS-CoV infection. In someembodiments, the antibody or antigen binding fragment includes a V_(H)and a V_(L) comprising the amino acid sequences set forth as SEQ ID NOs:127 and 129, respectively, and can specifically bind to MERS-CoV Sprotein and neutralize MERS-CoV infection.

1. Additional Description of Antibodies and Antigen Binding Fragments

The antibody or antigen binding fragment can be a human antibody orfragment thereof. Chimeric antibodies are also provided. The antibody orantigen binding fragment can include any suitable framework region, suchas (but not limited to) a human framework region. Human frameworkregions, and mutations that can be made in a human antibody frameworkregions, are known in the art (see, for example, in U.S. Pat. No.5,585,089, which is incorporated herein by reference). The frameworkregions of the JC57-13, JC57-14, and JC57-11 antibodies can be swappedfor human framework regions to generate a fully human antibody.Alternatively, a heterologous framework region, such as, but not limitedto a mouse framework region, can be included in the heavy or light chainof the antibodies. (See, for example, Jones et al., Nature 321:522,1986; Riechmann et al., Nature 332:323, 1988; Verhoeyen et al., Science239:1534, 1988; Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285,1992; Sandhu, Crit. Rev. Biotech. 12:437, 1992; and Singer et al., J.Immunol. 150:2844, 1993.)

The antibody can be of any isotype. The antibody can be, for example, anIgM or an IgG antibody, such as IgG₁, IgG₂, IgG₃, or IgG₄. The class ofan antibody that specifically binds MERS-CoV S protein can be switchedwith another. In one aspect, a nucleic acid molecule encoding V_(L) orV_(H) is isolated using methods well-known in the art, such that it doesnot include any nucleic acid sequences encoding the constant region ofthe light or heavy chain, respectively. A nucleic acid molecule encodingV_(L) or V_(H) is then operatively linked to a nucleic acid sequenceencoding a C_(L) or C_(H) from a different class of immunoglobulinmolecule. This can be achieved using a vector or nucleic acid moleculethat comprises a C_(L) or C_(H) chain, as known in the art. For example,an antibody that specifically binds MERS-CoV S protein, that wasoriginally IgM may be class switched to an IgG. Class switching can beused to convert one IgG subclass to another, such as from IgG₁ to IgG₂,IgG₃, or IgG₄.

In some examples, the disclosed antibodies are oligomers of antibodies,such as dimers, trimers, tetramers, pentamers, hexamers, septamers,octomers and so on.

(a) Binding Affinity

In several embodiments, the antibody or antigen binding fragment canspecifically bind MERS-CoV S protein with an affinity (e.g., measured byK_(d)) of less than 1.0×10⁻⁸M (such as less than 5.0×10⁻⁸M, less than1.0×10⁻⁹M, less than 5.0×10M, less than 1.0×10⁻¹° M, less than 5.0×10⁻¹°M, or less than 1.0×10⁻¹¹ M. K_(d) can be measured, for example, by aradiolabeled antigen binding assay (RIA) performed with the Fab versionof an antibody of interest and its antigen using known methods. In oneassay, solution binding affinity of Fabs for antigen is measured byequilibrating Fab with a minimal concentration of (¹²⁵I)-labeled antigenin the presence of a titration series of unlabeled antigen, thencapturing bound antigen with an anti-Fab antibody-coated plate (see,e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establishconditions for the assay, MICROTITER® multi-well plates (ThermoScientific) are coated overnight with 5 μg/ml of a capturing anti-Fabantibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), andsubsequently blocked with 2% (w/v) bovine serum albumin in PBS for twoto five hours at room temperature (approximately 23° C.). In anon-adsorbent plate (Nunc #269620), 100 μM or 26 μM [¹²⁵I]-antigen aremixed with serial dilutions of a Fab of interest (e.g., consistent withassessment of the anti-VEGF antibody, Fab-12, in Presta et al., CancerRes. 57:4593-4599 (1997)). The Fab of interest is then incubatedovernight; however, the incubation may continue for a longer period(e.g., about 65 hours) to ensure that equilibrium is reached.Thereafter, the mixtures are transferred to the capture plate forincubation at room temperature (e.g., for one hour). The solution isthen removed and the plate washed eight times with 0.1% polysorbate 20(TWEEN-20®) in PBS. When the plates have dried, 150 μl/well ofscintillant (MICROSCINT-20™; Packard) is added, and the plates arecounted on a TOPCOUNT™ gamma counter (Packard) for ten minutes.Concentrations of each Fab that give less than or equal to 20% ofmaximal binding are chosen for use in competitive binding assays.

In another assay, K_(d) can be measured using surface plasmon resonanceassays using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore, Inc.,Piscataway, N.J.) at 25° C. with immobilized antigen CMS chips at ˜10response units (RU). Briefly, carboxymethylated dextran biosensor chips(CMS, BIACORE®, Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (˜0.2μM) before injection at a flow rate of 5 l/minute to achieveapproximately 10 response units (RU) of coupled protein. Following theinjection of antigen, 1 M ethanolamine is injected to block unreactedgroups. For kinetics measurements, two-fold serial dilutions of Fab(0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20(TWEEN-20™) surfactant (PBST) at 25° C. at a flow rate of approximately25 l/min. Association rates (k_(on)) and dissociation rates (k_(off))are calculated using a simple one-to-one Langmuir binding model(BIACORE® Evaluation Software version 3.2) by simultaneously fitting theassociation and dissociation sensorgrams. The equilibrium dissociationconstant (Kd) is calculated as the ratio k_(off)/k_(on). See, e.g., Chenet al., J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 106 M⁻¹s⁻¹ by the surface plasmon resonance assay above, then the on-rate canbe determined by using a fluorescent quenching technique that measuresthe increase or decrease in fluorescence emission intensity(excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence ofincreasing concentrations of antigen as measured in a spectrometer, suchas a stop-flow equipped spectrophometer (Aviv Instruments) or a8000-series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with astirred cuvette.

(b) Neutralization

In some embodiments, the antibody or antigen binding fragment can alsobe distinguished by neutralization breadth. In some embodiments, theantibody or antigen binding fragment can neutralize at least 70% (suchas at least 75%, at least 80%, at least 85%, or least 90%,) of theMERS-CoV isolates listed Table 4, for example, with an IC50 value ofless than 50 μg/ml. The person of ordinary skill in the art if familiarwith methods of measuring neutralization breadth and potency.Additionally, exemplary methods for assaying the neutralization breadthand potency of an immune response to a vaccination regimen are providedherein in the Examples section.

(c) Multispecific Antibodies

In some embodiments, the antibody or antigen binding fragment isincluded on a multispecific antibody, such as a bi-specific antibody.Such multispecific antibodies can be produced by known methods, such ascrosslinking two or more antibodies, antigen binding fragments (such asscFvs) of the same type or of different types. Exemplary methods ofmaking multispecific antibodies include those described in PCT Pub. No.WO2013/163427, which is incorporated by reference herein in itsentirety. Suitable crosslinkers include those that areheterobifunctional, having two distinctly reactive groups separated byan appropriate spacer (such as m-maleimidobenzoyl-N-hydroxysuccinimideester) or homobifunctional (such as disuccinimidyl suberate). Suchlinkers are available from Pierce Chemical Company, Rockford, Ill.

Various types of multi-specific antibodies are known. Bispecific singlechain antibodies can be encoded by a single nucleic acid molecule.Examples of bispecific single chain antibodies, as well as methods ofconstructing such antibodies are known in the art (see, e.g., U.S. Pat.Nos. 8,076,459, 8,017,748, 8,007,796, 7,919,089, 7,820,166, 7,635,472,7,575,923, 7,435,549, 7,332,168, 7,323,440, 7,235,641, 7,229,760,7,112,324, 6,723,538, incorporated by reference herein). Additionalexamples of bispecific single chain antibodies can be found in PCTapplication No. WO 99/54440; Mack, J. Immunol., 158:3965-3970, 1997;Mack, PNAS, 92:7021-7025, 1995; Kufer, Cancer Immunol. Immunother.,45:193-197, 1997; Loffler, Blood, 95:2098-2103, 2000; and Bruhl, J.Immunol., 166:2420-2426, 2001. Production of bispecific Fab-scFv(“bibody”) molecules are described, for example, in Schoonjans et al.(J. Immunol. 165:7050-57, 2000) and Willems et al. (J Chromatogr BAnalyt Technol Biomed Life Sci. 786:161-76, 2003). For bibodies, a scFvmolecule can be fused to one of the VL-CL (L) or VH—CH1 chains, e.g., toproduce a bibody one scFv is fused to the C-term of a Fab chain.

(d) Fragments

Antigen binding fragments are encompassed by the present disclosure,such as Fab, F(ab′)₂, and Fv which include a heavy chain and light chainvariable region and specifically bind MERS-CoV S protein. These antibodyfragments retain the ability to selectively bind with the antigen andare “antigen-binding” fragments. These fragments include:

(1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule, can be produced by digestion of wholeantibody with the enzyme papain to yield an intact light chain and aportion of one heavy chain;

(2) Fab′, the fragment of an antibody molecule can be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain; two Fab′ fragmentsare obtained per antibody molecule;

(3) (Fab′)₂, the fragment of the antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction; F(ab′)₂ is a dimer of two Fab′ fragments held together by twodisulfide bonds;

(4) Fv, a genetically engineered fragment containing the variable regionof the light chain and the variable region of the heavy chain expressedas two chains; and

(5) Single chain antibody (such as scFv), defined as a geneticallyengineered molecule containing the variable region of the light chain,the variable region of the heavy chain, linked by a suitable polypeptidelinker as a genetically fused single chain molecule. A scFv is a fusionprotein in which a light chain variable region of an immunoglobulin anda heavy chain variable region of an immunoglobulin are bound by a linker(see, e.g., Ahmad et al., Clin. Dev. Immunol., 2012,doi:10.1155/2012/980250; Marbry, IDrugs, 13:543-549, 2010). Theintramolecular orientation of the V_(H)-domain and the V_(L)-domain in ascFv, is not decisive for the provided antibodies (e.g., for theprovided multispecific antibodies). Thus, scFvs with both possiblearrangements (V_(H)-domain-linker domain-V_(L)-domain;V_(L)-domain-linker domain-V_(H)-domain) may be used.

(6) A dimer of a single chain antibody (scFV₂), defined as a dimer of ascFV. This has also been termed a “miniantibody.”

Methods of making these fragments are known in the art (see for example,Harlow and Lane, Antibodies: A Laboratory Manual, 2^(nd), Cold SpringHarbor Laboratory, New York, 2013).

Antigen binding fragments can be prepared by proteolytic hydrolysis ofthe antibody or by expression in a host cell (such as an E. coli cell)of DNA encoding the fragment. Antigen binding fragments can also beobtained by pepsin or papain digestion of whole antibodies byconventional methods. For example, antigen binding fragments can beproduced by enzymatic cleavage of antibodies with pepsin to provide a 5Sfragment denoted F(ab′)₂. This fragment can be further cleaved using athiol reducing agent, and optionally a blocking group for the sulfhydrylgroups resulting from cleavage of disulfide linkages, to produce 3.5SFab′ monovalent fragments. Alternatively, an enzymatic cleavage usingpepsin produces two monovalent Fab′ fragments and an Fc fragmentdirectly (see U.S. Pat. Nos. 4,036,945 and 4,331,647, and referencescontained therein; Nisonhoff et al., Arch. Biochem. Biophys. 89:230,1960; Porter, Biochem. J. 73:119, 1959; Edelman et al., Methods inEnzymology, Vol. 1, page 422, Academic Press, 1967; and Coligan et al.at sections 2.8.1-2.8.10 and 2.10.1-2.10.4).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

Antigen binding single V_(H) domains, called domain antibodies (dAb),have also been identified from a library of murine V_(H) genes amplifiedfrom genomic DNA of immunized mice (Ward et al. Nature 341:544-546,1989). Human single immunoglobulin variable domain polypeptides capableof binding antigen with high affinity have also been described (see, forexample, PCT Publication Nos. WO 2005/035572 and WO 2003/002609). TheCDRs disclosed herein can also be included in a dAb.

In some embodiments, one or more of the heavy and/or light chaincomplementarity determining regions (CDRs) from a disclosed antibody(such as the JC57-13, JC57-11, JC57-14, FIB_B2, FIB_H1, G2, G4, D12,F11, C2, C5, A2, or A10 antibody) is expressed on the surface of anotherprotein, such as a scaffold protein. The expression of domains ofantibodies on the surface of a scaffolding protein are known in the art(see e.g. Liu et al., J. Virology 85(17): 8467-8476, 2011). Suchexpression creates a chimeric protein that retains the binding forMERS-CoV S protein. In some specific embodiments, one or more of theheavy chain CDRs is grafted onto a scaffold protein, such as one or moreof heavy chain CDR1, CDR2, and/or CDR3. One or more CDRs can also beincluded in a diabody or another type of single chain antibody molecule.

In an additional embodiment, the antibody fragment is a camelidantibody, and includes a heavy chain variable domain, but not a lightchain variable domain, of a disclosed antibody. Antibody proteinsobtained from members of the camel and dromedary (Camelus bactrianus andCalelus dromaderius) family including new world members such as llamaspecies (Lama paccos, Lama glama and Lama vicugna) have beencharacterized with respect to size, structural complexity andantigenicity for human subjects. Certain IgG antibodies from this familyof mammals as found in nature lack light chains, and are thusstructurally distinct from the typical four chain quaternary structurehaving two heavy and two light chains, for antibodies from otheranimals. See PCT/EP93/02214 (WO 94/04678 published 3 Mar. 1994).

A region of the camelid antibody which is the small single variabledomain identified as VHH can be obtained by genetic engineering to yielda small protein having high affinity for a target, resulting in a lowmolecular weight antibody-derived protein known as a “camelid nanobody”.See U.S. Pat. No. 5,759,808 issued Jun. 2, 1998; see also Stijlemans, B.et al., 2004 J Biol Chem 279: 1256-1261; Dumoulin, M. et al., 2003Nature 424: 783-788; Pleschberger, M. et al. 2003 Bioconjugate Chem 14:440-448; Cortez-Retamozo, V. et al. 2002 Int J Cancer 89: 456-62; andLauwereys, M. et al. 1998 EMBO J 17: 3512-3520. Engineered libraries ofcamelid antibodies and antibody fragments are commercially available,for example, from Ablynx, Ghent, Belgium. As with other antibodies ofnon-human origin, an amino acid sequence of a camelid antibody can bealtered recombinantly to obtain a sequence that more closely resembles ahuman sequence, i.e., the nanobody can be “humanized”. Thus the naturallow antigenicity of camelid antibodies to humans can be further reduced.The camelid nanobody has a molecular weight approximately one-tenth thatof a human IgG molecule, and the protein has a physical diameter of onlya few nanometers. One consequence of the small size is the ability ofcamelid nanobodies to bind to antigenic sites that are functionallyinvisible to larger antibody proteins, i.e., camelid nanobodies areuseful as reagents detect antigens that are otherwise cryptic usingclassical immunological techniques, and as possible therapeutic agents.Thus yet another consequence of small size is that a camelid nanobodycan inhibit as a result of binding to a specific site in a groove ornarrow cleft of a target protein, and hence can serve in a capacity thatmore closely resembles the function of a classical low molecular weightdrug than that of a classical antibody.

The low molecular weight and compact size further result in camelidnanobodies being extremely thermostable, stable to extreme pH and toproteolytic digestion, and poorly antigenic. Another consequence is thatcamelid nanobodies readily move from the circulatory system intotissues, and even cross the blood-brain barrier and can treat disordersthat affect nervous tissue. Nanobodies can further facilitated drugtransport across the blood brain barrier. See U.S. patent application20040161738 published Aug. 19, 2004. These features combined with thelow antigenicity to humans indicate great therapeutic potential.Further, these molecules can be fully expressed in prokaryotic cellssuch as E. coli and are expressed as fusion proteins with bacteriophageand are functional.

Accordingly, a feature of the present invention is a camelid antibody ornanobody having high affinity for MERS-CoV S protein. In severalembodiment, the camelid antibody or nanobody is obtained by grafting theCDRs sequences of the heavy or light chain of the disclosed MERS-CoV Sprotein specific antibodies into nanobody or single domain antibodyframework sequences, as described for example in PCT/EP93/02214.

(e) Variants

In certain embodiments, amino acid sequence variants of the antibodiesprovided herein are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody. Amino acid sequence variants of an antibody may be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the antibody, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., antigen-binding.

In certain embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the CDRs and the framework regions. Amino acidsubstitutions may be introduced into an antibody of interest and theproducts screened for a desired activity, e.g., retained/improvedantigen binding, decreased immunogenicity, or improved ADCC or CDC.

The variants typically retain amino acid residues necessary for correctfolding and stabilizing between the V_(H) and the V_(L) regions, andwill retain the charge characteristics of the residues in order topreserve the low pI and low toxicity of the molecules. Amino acidsubstitutions can be made in the V_(H) and the V_(L) regions to increaseyield. Conservative amino acid substitution tables providingfunctionally similar amino acids are well known to one of ordinary skillin the art.

In some embodiments, the V_(H) of the antibody includes up to 10 (suchas up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to8, or up to 9) amino acid substitutions (such as conservative amino acidsubstitutions), and or the V_(L) of the antibody includes up to 10 (suchas up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to8, or up to 9) amino acid substitutions (such as conservative amino acidsubstitutions), compared to the amino acid sequence of the correspondingV_(H) and/or V_(L) of one of the JC57-13, JC57-11, JC57-14, FIB_B2,FIB_H1, G2, G4, D12, F11, C2, C5, A2, or A10 antibodies.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more CDRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in CDRs. In certain embodiments of the variant VH and VLsequences provided above, each CDR either is unaltered, or contains nomore than one, two or three amino acid substitutions.

To increase binding affinity of the antibody, the V_(L) and V_(H)segments can be randomly mutated, such as within HCDR3 region or theLCDR3 region, in a process analogous to the in vivo somatic mutationprocess responsible for affinity maturation of antibodies during anatural immune response. Thus in vitro affinity maturation can beaccomplished by amplifying V_(H) and V_(L) regions using PCR primerscomplementary to the HCDR3 or LCDR3, respectively. In this process, theprimers have been “spiked” with a random mixture of the four nucleotidebases at certain positions such that the resultant PCR products encodeV_(H) and V_(L) segments into which random mutations have beenintroduced into the V_(H) and/or V_(L) CDR3 regions. These randomlymutated V_(H) and V_(L) segments can be tested to determine the bindingaffinity for MERS-CoV S protein. Methods of in vitro affinity maturationare known (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)),and Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brienet al., ed., Human Press, Totowa, N.J., (2001).)

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham and Wells (1989) Science,244:1081-1085. In this method, a residue or group of target residues(e.g., charged residues such as arg, asp, his, lys, and glu) areidentified and replaced by a neutral or negatively charged amino acid(e.g., alanine or polyalanine) to determine whether the interaction ofthe antibody with antigen is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an antigen-antibody complex is usedto identify contact points between the antibody and antigen. Suchcontact residues and neighboring residues may be targeted or eliminatedas candidates for substitution. Variants may be screened to determinewhether they contain the desired properties.

In certain embodiments, an antibody or antigen binding fragment isaltered to increase or decrease the extent to which the antibody orantigen binding fragment is glycosylated. Addition or deletion ofglycosylation sites may be conveniently accomplished by altering theamino acid sequence such that one or more glycosylation sites is createdor removed.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH₂ domain of the Fcregion. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody may be made in order to create antibodyvariants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydratestructure that lacks fucose attached (directly or indirectly) to an Fcregion. For example, the amount of fucose in such antibody may be from1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amountof fucose is determined by calculating the average amount of fucosewithin the sugar chain at Asn297, relative to the sum of allglycostructures attached to Asn 297 (e.g. complex, hybrid and highmannose structures) as measured by MALDI-TOF mass spectrometry, asdescribed in WO 2008/077546, for example. Asn297 refers to theasparagine residue located at about position 297 in the Fc region;however, Asn297 may also be located about ±3 amino acids upstream ordownstream of position 297, i.e., between positions 294 and 300, due tominor sequence variations in antibodies. Such fucosylation variants mayhave improved ADCC function. See, e.g., US Patent Publication Nos. US2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd).Examples of publications related to “defucosylated” or“fucose-deficient” antibody variants include: US 2003/0157108; WO2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO2005/035778; WO2005/053742; WO2002/031140; Okazaki et al. J. Mol. Biol.336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614(2004). Examples of cell lines capable of producing defucosylatedantibodies include Lec 13 CHO cells deficient in protein fucosylation(Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl NoUS 2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al.,especially at Example 11), and knockout cell lines, such asalpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g.,Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al.,Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Antibodies variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet etal.); U.S. Pat. No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umanaet al.). Antibody variants with at least one galactose residue in theoligosaccharide attached to the Fc region are also provided. Suchantibody variants may have improved CDC function. Such antibody variantsare described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964(Raju, S.); and WO 1999/22764 (Raju, S.).

In several embodiments, the constant region of the antibody includes oneor more amino acid substitutions to optimize in vivo half-life of theantibody. The serum half-life of IgG Abs is regulated by the neonatal Fcreceptor (FcRn). Thus, in several embodiments, the antibody includes anamino acid substitution that increases binding to the FcRn. Several suchsubstitutions are known to the person of ordinary skill in the art, suchas substitutions at IgG constant regions T250Q and M428L (see, e.g.,Hinton et al., J ImmunoL, 176:346-356, 2006); M428L and N434S (see,e.g., Zalevsky, et al., Nature Biotechnology, 28:157-159, 2010); N434A(see, e.g., Petkova et al., Int. Immunol., 18:1759-1769, 2006); T307A,E380A, and N434A (see, e.g., Petkova et al., Int. Immunol.,18:1759-1769, 2006); and M252Y, S254T, and T256E (see, e.g., Dall'Acquaet al., J. Biol. Chem., 281:23514-23524, 2006).

In some embodiments, the constant region of the antibody includes one ofmore amino acid substitutions to optimize Antibody-dependentcell-mediated cytotoxicity (ADCC). ADCC is mediated primarily through aset of closely related Fcγ receptors. In some embodiments, the antibodyincludes one or more amino acid substitutions that increase binding toFcγRIIIa. Several such substitutions are known to the person of ordinaryskill in the art, such as substitutions at IgG constant regions S239Dand I332E (see, e.g., Lazar et al., Proc. Natl., Acad. Sci. U.S.A.,103:4005-4010, 2006); and S239D, A330L, and I332E (see, e.g., Lazar etal., Proc. Natl., Acad. Sci. U.S.A., 103:4005-4010, 2006).

Combinations of the above substitutions are also included, to generatean IgG constant region with increased binding to FcRn and FcγRIIIa. Thecombinations increase antibody half-life and ADCC. For example, suchcombination include antibodies with the following amino acidsubstitution in the Fc region:

(1) S239D/I332E and T250Q/M428L;

(2) S239D/I332E and M428L/N434S;

(3) S239D/I332E and N434A;

(4) S239D/I332E and T307A/E380A/N434A;

(5) S239D/I332E and M252Y/S254T/T256E;

(6) S239D/A330L/1332E and T250Q/M428L;

(7) S239D/A330L/1332E and M428L/N434S;

(8) S239D/A330L/1332E and N434A;

(9) S239D/A330L/1332E and T307A/E380A/N434A; or

(10) S239D/A330L/1332E and M252Y/S254T/T256E.

In some examples, the antibodies, or an antigen binding fragment thereofis modified such that it is directly cytotoxic to infected cells, oruses natural defenses such as complement, antibody dependent cellularcytotoxicity (ADCC), or phagocytosis by macrophages.

In certain embodiments, an antibody provided herein may be furthermodified to contain additional nonproteinaceous moieties that are knownin the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer areattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,etc.

The antibody or antigen binding fragment can be derivatized or linked toanother molecule (such as another peptide or protein). In general, theantibody or antigen binding fragment is derivatized such that thebinding to MERS-CoV S protein is not affected adversely by thederivatization or labeling. For example, the antibody or antigen bindingfragment can be functionally linked (by chemical coupling, geneticfusion, noncovalent association or otherwise) to one or more othermolecular entities, such as another antibody (for example, a bi-specificantibody or a diabody), a detectable marker, an effector molecule, or aprotein or peptide that can mediate association of the antibody orantibody portion with another molecule (such as a streptavidin coreregion or a polyhistidine tag).

B. Conjugates

Monoclonal antibodies and antigen binding fragments that specificallybind to an epitope on MERS-CoV S protein can be conjugated to an agent,such as an effector molecule or detectable marker, using any number ofmeans known to those of skill in the art. Both covalent and noncovalentattachment means may be used. One of skill in the art will appreciatethat various effector molecules and detectable markers can be used,including (but not limited to) toxins and radioactive agents such as¹²⁵I, ³²P, ³H and ³⁵S and other labels, target moieties and ligands,etc. The choice of a particular effector molecule or detectable markerdepends on the particular target molecule or cell, and the desiredbiological effect.

The choice of a particular effector molecule or detectable markerdepends on the particular target molecule or cell, and the desiredbiological effect. Thus, for example, the effector molecule can be acytotoxin that is used to bring about the death of a particular targetcell (such as a MERS-CoV infected cell). In other embodiments, theeffector molecule can be a cytokine, such as IL-15; conjugates includingthe cytokine can be used, e.g., to stimulate immune calls locally.

The procedure for attaching an effector molecule or detectable marker toan antibody or antigen binding fragment varies according to the chemicalstructure of the effector. Polypeptides typically contain a variety offunctional groups; such as carboxylic acid (COOH), free amine (—NH₂) orsulfhydryl (—SH) groups, which are available for reaction with asuitable functional group on a polypeptide to result in the binding ofthe effector molecule or detectable marker. Alternatively, the antibodyor antigen binding fragment is derivatized to expose or attachadditional reactive functional groups. The derivatization may involveattachment of any of a number of known linker molecules such as thoseavailable from Pierce Chemical Company, Rockford, Ill. The linker can beany molecule used to join the antibody or antigen binding fragment tothe effector molecule or detectable marker. The linker is capable offorming covalent bonds to both the antibody or antigen binding fragmentand to the effector molecule or detectable marker. Suitable linkers arewell known to those of skill in the art and include, but are not limitedto, straight or branched-chain carbon linkers, heterocyclic carbonlinkers, or peptide linkers. Where the antibody or antigen bindingfragment and the effector molecule or detectable marker arepolypeptides, the linkers may be joined to the constituent amino acidsthrough their side groups (such as through a disulfide linkage tocysteine) or to the alpha carbon amino and carboxyl groups of theterminal amino acids.

In several embodiments, the linker can include a spacer element, which,when present, increases the size of the linker such that the distancebetween the effector molecule or the detectable marker and the antibodyor antigen binding fragment is increased. Exemplary spacers are known tothe person of ordinary skill, and include those listed in U.S. Pat. Nos.7,964,5667, 498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065,5,780,588, 5,665,860, 5,663,149, 5,635,483, 5,599,902, 5,554,725,5,530,097, 5,521,284, 5,504,191, 5,410,024, 5,138,036, 5,076,973,4,986,988, 4,978,744, 4,879,278, 4,816,444, and 4,486,414, as well asU.S. Pat. Pub. Nos. 20110212088 and 20110070248, each of which isincorporated by reference in its entirety.

In some embodiments, the linker is cleavable under intracellularconditions, such that cleavage of the linker releases the effectormolecule or detectable marker from the antibody or antigen bindingfragment in the intracellular environment. In yet other embodiments, thelinker is not cleavable and the effector molecule or detectable markeris released, for example, by antibody degradation. In some embodiments,the linker is cleavable by a cleaving agent that is present in theintracellular environment (for example, within a lysosome or endosome orcaveolea). The linker can be, for example, a peptide linker that iscleaved by an intracellular peptidase or protease enzyme, including, butnot limited to, a lysosomal or endosomal protease. In some embodiments,the peptide linker is at least two amino acids long or at least threeamino acids long. However, the linker can be 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14 or 15 amino acids long, such as 1-2, 1-3, 2-5, 3-10, 3-15,1-5, 1-10, 1-15, amino acids long.

In view of the large number of methods that have been reported forattaching a variety of radiodiagnostic compounds, radiotherapeuticcompounds, labels (such as enzymes or fluorescent molecules), toxins,and other agents to antibodies one skilled in the art will be able todetermine a suitable method for attaching a given agent to an antibodyor antigen binding fragment or other polypeptide. For example, theantibody or antigen binding fragment can be conjugated with effectormolecules such as small molecular weight drugs such as MonomethylAuristatin E (MMAE), Monomethyl Auristatin F (MMAF), maytansine,maytansine derivatives, including the derivative of maytansine known asDM1 (also known as mertansine), or other agents to make an antibody drugconjugate (ADC). In several embodiments, conjugates of an antibody orantigen binding fragment and one or more small molecule toxins, such asa calicheamicin, maytansinoids, dolastatins, auristatins, atrichothecene, and CC1065, and the derivatives of these toxins that havetoxin activity, are provided.

The antibody or antigen binding fragment can be conjugated with adetectable marker; for example, a detectable marker capable of detectionby ELISA, spectrophotometry, flow cytometry, microscopy or diagnosticimaging techniques (such as computed tomography (CT), computed axialtomography (CAT) scans, magnetic resonance imaging (MRI), nuclearmagnetic resonance imaging NMRI), magnetic resonance tomography (MTR),ultrasound, fiberoptic examination, and laparoscopic examination).Specific, non-limiting examples of detectable markers includefluorophores, chemiluminescent agents, enzymatic linkages, radioactiveisotopes and heavy metals or compounds (for example super paramagneticiron oxide nanocrystals for detection by MRI). For example, usefuldetectable markers include fluorescent compounds, including fluorescein,fluorescein isothiocyanate, rhodamine,5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin, lanthanidephosphors and the like. Bioluminescent markers are also of use, such asluciferase, Green fluorescent protein (GFP), Yellow fluorescent protein(YFP). An antibody or antigen binding fragment can also be conjugatedwith enzymes that are useful for detection, such as horseradishperoxidase, β-galactosidase, luciferase, alkaline phosphatase, glucoseoxidase and the like. When an antibody or antigen binding fragment isconjugated with a detectable enzyme, it can be detected by addingadditional reagents that the enzyme uses to produce a reaction productthat can be discerned. For example, when the agent horseradishperoxidase is present the addition of hydrogen peroxide anddiaminobenzidine leads to a colored reaction product, which is visuallydetectable. An antibody or antigen binding fragment may also beconjugated with biotin, and detected through indirect measurement ofavidin or streptavidin binding. It should be noted that the avidinitself can be conjugated with an enzyme or a fluorescent label.

The antibody or antigen binding fragment can be conjugated with aparamagnetic agent, such as gadolinium. Paramagnetic agents such assuperparamagnetic iron oxide are also of use as labels. Antibodies canalso be conjugated with lanthanides (such as europium and dysprosium),and manganese. An antibody or antigen binding fragment may also belabeled with a predetermined polypeptide epitopes recognized by asecondary reporter (such as leucine zipper pair sequences, binding sitesfor secondary antibodies, metal binding domains, epitope tags).

The antibody or antigen binding fragment can also be conjugated with aradiolabeled amino acid. The radiolabel may be used for both diagnosticand therapeutic purposes. For instance, the radiolabel may be used todetect MERS-CoV S protein and MERS-CoV S protein expressing cells byx-ray, emission spectra, or other diagnostic techniques. Examples oflabels for polypeptides include, but are not limited to, the followingradioisotopes or radionucleotides: ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In,¹²⁵I, ¹³¹I.

Means of detecting such detectable markers are well known to those ofskill in the art. Thus, for example, radiolabels may be detected usingphotographic film or scintillation counters, fluorescent markers may bedetected using a photodetector to detect emitted illumination. Enzymaticlabels are typically detected by providing the enzyme with a substrateand detecting the reaction product produced by the action of the enzymeon the substrate, and colorimetric labels are detected by simplyvisualizing the colored label.

The average number of effector molecule or detectable marker moietiesper antibody or antigen binding fragment in a conjugate can range, forexample, from 1 to 20 moieties per antibody or antigen binding fragment.In certain embodiments, the average number of effector molecule ordetectable marker moieties per antibody or antigen binding fragment in aconjugate range from about 1 to about 2, from about 1 to about 3, about1 to about 8; from about 2 to about 6; from about 3 to about 5; or fromabout 3 to about 4. The loading (for example, effector molecule/antibodyratio) of an conjugate may be controlled in different ways, for example,by: (i) limiting the molar excess of effector molecule-linkerintermediate or linker reagent relative to antibody, (ii) limiting theconjugation reaction time or temperature, (iii) partial or limitingreductive conditions for cysteine thiol modification, (iv) engineeringby recombinant techniques the amino acid sequence of the antibody suchthat the number and position of cysteine residues is modified forcontrol of the number or position of linker-effector moleculeattachments.

C. Methods

Methods are disclosed herein for the prevention or treatment of MERS-CoVinfection in a subject by administering a therapeutically effectiveamount of a disclosed MERS-CoV S protein specific antibody or antigenbinding fragment, or encoding nucleic acid molecule, to the subject. Insome examples, the antibody, antigen binding fragment, or nucleic acidmolecule, can be used pre-exposure (for example, to prevent or inhibitMERS-CoV infection). In some examples, the antibody, antigen bindingfragment, or nucleic acid molecule, can be used in post-exposureprophylaxis. In some examples, the antibody, antigen binding fragment,or nucleic acid molecule, can be administered to a subject with aMERS-CoV infection, such as a subject being treated with anti-viraltherapy. In some examples the antibody, antigen binding fragment, ornucleic acid molecule is modified such that it is directly cytotoxic toinfected cells (e.g., by conjugation to a toxin), or uses naturaldefenses such as complement, antibody dependent cellular cytotoxicity(ADCC), or phagocytosis by macrophages.

MERS-CoV infection does not need to be completely eliminated orprevented for the method to be effective. For example, a method candecrease MERS-CoV infection in the subject by a desired amount, forexample by at least 10%, at least 20%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98%, oreven at least 100% (elimination of detectable MERS-CoV infected cells),as compared to MERS-CoV infection in the absence of the treatment. Inadditional embodiments, MERS-CoV replication can be reduced or inhibitedby similar methods. MERS-CoV replication does not need to be completelyeliminated for the method to be effective. For example, a method candecrease MERS-CoV replication by a desired amount, for example by atleast 10%, at least 20%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, at least 98%, or even at least100% (elimination of detectable MERS-CoV), as compared to MERS-CoVreplication in the absence of the treatment.

In one embodiment, administration of a disclosed antibody, antigenbinding fragment, or conjugate, or nucleic acid molecule, results in areduction in the establishment of MERS-CoV infection and/or reducingsubsequent MERS-CoV disease progression in a subject. A reduction in theestablishment of MERS-CoV infection and/or a reduction in subsequentMERS-CoV disease progression encompass any statistically significantreduction in MERS-CoV activity.

For any application, the antibody, antigen binding fragment, orconjugate, or nucleic acid molecule can be combined with anti-viraltherapy.

A therapeutically effective amount of a MERS-CoV S protein-specificantibody, antigen binding fragment, or conjugate, or nucleic acidmolecule encoding such molecules, will depend upon the severity of thedisease and/or infection and the general state of the patient's health.A therapeutically effective amount is that which provides eithersubjective relief of a symptom(s) or an objectively identifiableimprovement as noted by the clinician or other qualified observer. TheMERS-CoV S protein-specific antibody, antigen binding fragment, orconjugate, or nucleic acid molecule encoding such molecules, can beadministered in conjunction with another therapeutic agent, eithersimultaneously or sequentially.

Single or multiple administrations of a composition including adisclosed MERS-CoV S protein-specific antibody, antigen bindingfragment, or conjugate, or nucleic acid molecule encoding suchmolecules, can be administered depending on the dosage and frequency asrequired and tolerated by the patient. Compositions including theMERS-CoV S protein-specific antibody, antigen binding fragment, orconjugate, or nucleic acid molecule encoding such molecules, shouldprovide a sufficient quantity of at least one of the MERS-CoV Sprotein-specific antibodies, antigen binding fragments, or conjugates,or nucleic acid molecule encoding such molecules to effectively treatthe patient. The dosage can be administered once, but may be appliedperiodically until either a therapeutic result is achieved or until sideeffects warrant discontinuation of therapy. In one example, a dose ofthe antibody or antigen binding fragment is infused for thirty minutesevery other day. In this example, about one to about ten doses can beadministered, such as three or six doses can be administered every otherday. In a further example, a continuous infusion is administered forabout five to about ten days. The subject can be treated at regularintervals, such as monthly, until a desired therapeutic result isachieved. Generally, the dose is sufficient to treat or amelioratesymptoms or signs of disease without producing unacceptable toxicity tothe patient.

Data obtained from cell culture assays and animal studies can be used toformulate a range of dosage for use in humans. The dosage normally lieswithin a range of circulating concentrations that include the ED₅₀, withlittle or minimal toxicity. The dosage can vary within this rangedepending upon the dosage form employed and the route of administrationutilized. The therapeutically effective dose can be determined from cellculture assays and animal studies.

In certain embodiments, the antibody or antigen binding fragment thatspecifically binds MERS-CoV S protein, or conjugate thereof, or anucleic acid molecule or vector encoding such a molecule, or acomposition including such molecules, is administered at a dose in therange of from about 5 or 10 nmol/kg to about 300 nmol/kg, or from about20 nmol/kg to about 200 nmol/kg, or at a dose of about 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110,120, 125, 130, 140, 150, 160, 170, 175, 180, 190, 200, 210, 220, 230,240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 750, 1000, 1250,1500, 1750 or 2000 nmol/kg, or at a dose of about 5, 10, 20, 30, 40, 50,60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,950 or 1000 μg/kg, or about 1, 1.25, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5,5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 mg/kg, or other dose deemedappropriate by the treating physician. The doses described herein can beadministered according to the dosing frequency/frequency ofadministration described herein, including without limitation daily, 2or 3 times per week, weekly, every 2 weeks, every 3 weeks, monthly, etc.

In some embodiments, a disclosed therapeutic agent may be administeredintravenously, subcutaneously or by another mode daily or multiple timesper week for a period of time, followed by a period of no treatment,then the cycle is repeated. The initial period of treatment (e.g.,administration of the therapeutic agent daily or multiple times perweek) can be for 1 day, 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks or 12weeks, for example. In a related embodiment, the period of no treatmentcan be for a period of days, weeks, or months, such as 3 days, 1 week, 2weeks, 3 weeks or 4 weeks, 2 months, 3 months, 4 months, 5 months, 6months, or more time. In certain embodiments, the dosing regimen of thetherapeutic agent is daily for 3 days followed by 3 days off; or dailyor multiple times per week for 1 week followed by 3 days or 1 week off;or daily or multiple times per week for 2 weeks followed by 1 or 2 weeksoff; or daily or multiple times per week for 3 weeks followed by 1, 2 or3 weeks off; or daily or multiple times per week for 4, 5, 6, 7, 8, 9,10, 11 or 12 weeks followed by 1, 2, 3 or 4 weeks off.

Methods are also provided for the detection of the expression ofMERS-CoV S protein in vitro or in vivo. In one example, the presence ofMERS-CoV S protein can be detected in a biological sample using adisclosed MERS-CoV S protein specific antibody or antigen bindingfragment thereof. Detecting the presence of the MERS-CoV S protein inthe biological sample indicates that the sample is from a subject with aMERS-CoV infection. The sample can be any sample, including, but notlimited to, tissue from biopsies, autopsies and pathology specimens.Biological samples also include sections of tissues, for example, frozensections taken for histological purposes. Biological samples furtherinclude body fluids, such as blood, serum, plasma, sputum, spinal fluidor urine. The method of detection can include contacting a cell orsample, or administering to a subject, an antibody or antigen bindingfragment that specifically binds to MERS-CoV S protein, or conjugatethere of (e.g. a conjugate including a detectable marker) underconditions sufficient to form an immune complex, and detecting theimmune complex (e.g., by detecting a detectable marker conjugated to theantibody or antigen binding fragment.

In several embodiments, a method is provided for detecting MERS-CoVinfection in a subject. The disclosure provides a method for detectingMERS-CoV in a biological sample, wherein the method includes contactinga biological sample from a subject with a disclosed antibody or antigenbinding fragment under conditions sufficient for formation of an immunecomplex, and detecting the immune complex, to detect the MERS-CoV Sprotein in the biological sample. In one example, the detection ofMERS-CoV S protein in the sample indicates that the subject has anMERS-CoV infection.

In some embodiments, the disclosed antibodies or antigen bindingfragments thereof are used to test vaccines. For example to test if avaccine composition including MERS-CoV S protein includes an epitopethat can be specifically bound by an antibody that neutralizes MERS-CoVinfection. Thus provided herein is a method for testing a vaccine,wherein the method includes contacting a sample containing the vaccine,such as a MERS-CoV S protein immunogen, with a disclosed antibody orantigen binding fragment under conditions sufficient for formation of animmune complex, and detecting the immune complex, to detect the vaccinewith epitope that can be specifically bound by an antibody thatneutralizes MERS-CoV infection in the sample.

In one embodiment, the antibody or antigen binding fragment is directlylabeled with a detectable marker. In another embodiment, the antibodythat binds MERS-CoV S protein (the first antibody) is unlabeled and asecond antibody or other molecule that can bind the antibody that bindsthe first antibody is utilized for detection. As is well known to one ofskill in the art, a second antibody is chosen that is able tospecifically bind the specific species and class of the first antibody.For example, if the first antibody is a human IgG, then the secondaryantibody may be an anti-human-IgG. Other molecules that can bind toantibodies include, without limitation, Protein A and Protein G, both ofwhich are available commercially.

Suitable labels for the antibody, antigen binding fragment or secondaryantibody are described above, and include various enzymes, prostheticgroups, fluorescent materials, luminescent materials, magnetic agentsand radioactive materials. Non-limiting examples of suitable enzymesinclude horseradish peroxidase, alkaline phosphatase,beta-galactosidase, or acetylcholinesterase. Non-limiting examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin. Non-limiting examples of suitable fluorescent materialsinclude umbelliferone, fluorescein, fluorescein isothiocyanate,rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride orphycoerythrin. A non-limiting exemplary luminescent material is luminol;a non-limiting exemplary a magnetic agent is gadolinium, andnon-limiting exemplary radioactive labels include ¹²⁵I, ¹³¹I, ³⁵S or ³H.

III. MERS-CoV Immunogens and their Use A. MERS-CoV S Proteins andImmunogenic Fragments Thereof

Several embodiments concern the MERS-CoV S protein or fragment orvariant thereof, or a nucleic acid molecule encoding such proteins. Aclass I fusion glycoprotein, the MERS-CoV S protein is initiallysynthesized as a precursor protein of approximately 1350 amino acids insize. Individual precursor S polypeptides form a homotrimer and undergoglycosylation within the Golgi apparatus as well as processing to removethe signal peptide, and cleavage by a cellular protease betweenapproximately position 751/752 to generate separate S1 and S2polypeptide chains, which remain associated as S1/S2 protomers withinthe homotrimer, providing a trimer of herterodimers. There is anothercleavage event that occurs between 887/888 to expose the N-terminus ofS2 and liberate the fusion peptide. The S1 subunit is distal to thevirus membrane and contains the receptor-binding domain (RBD) thatmediates virus attachment to its host receptor, dipeptidyl peptidase-4(DPP4), and includes approximately residues 367-606 of the S protein.The S2 subunit contains a transmembrane domain and two heptad-repeatsequences typical of fusion glycoproteins and mediates viral entry viamembrane fusion with the host target cell.

The numbering used in the disclosed MERS-CoV S proteins and fragmentsthereof is relative to the S protein of the England1 strain of MERS-CoV,the sequence of which is provided as SEQ ID NO: 14, and deposited inGenBank as No. AFY13307.1, which is incorporated by reference herein inits entirety. Additional strains of MERS-CoV are known. An exemplarynucleic acid sequence encoding full-length MERS-CoV S protein isprovided as SEQ ID NO: 13, below.

Full-length MERS-CoV S protein (1-1353) encoding sequence, England1strain (SEQ ID NO: 13)atgattcactccgtgttcctgctgatgttcctgctgactcctacagagagctatgtggatgtgggacctgattccgtcaagagcgcctgcatcgaagtggacattcagcagaccttctttgataagacatggccaagacccatcgacgtgagcaaagccgatggcatcatctaccctcaggggaggacctattccaatatcacaattacttaccagggcctgttcccatatcagggagaccacggcgatatgtacgtgtattctgctggccatgcaacagggaccacacctcagaagctgtttgtggctaactacagccaggacgtcaaacagttcgcaaatggatttgtggtccgcatcggcgccgctgcaaactctaccggcacagtgatcatttcacctagcacttccgcaaccatccgaaaaatctacccagccttcatgctgggaagctccgtgggcaattttagcgacgggaaaatgggacggttctttaaccacaccctggtgctgctgcctgatggatgcggcacactgctgagggctttctactgtatcctggagccacgcagcggaaaccactgccccgcaggaaatagctacacctcctttgccacatatcatactccagctaccgactgttccgatggcaactacaatcgaaacgcctctctgaatagtttcaaggaatacttcaacctgcggaattgcacattcatgtacacttataacatcaccgaggacgaaattctggagtggttcggaatcactcagaccgcacagggcgtgcacctgttttctagtcgctacgtcgacctgtatggcgggaacatgttccagtttgccactctgcccgtgtacgataccatcaagtactattccatcattcctcattcaatccgcagcattcagtccgatcgaaaggcttgggccgctttctacgtgtataaactgcagccactgaccttcctgctggactttagcgtcgatggctacatccggagagccattgactgcgggtttaatgatctgtcccagctgcactgttcttacgaaagtttcgacgtggagtccggcgtgtattctgtctcaagctttgaggccaagccctctgggagtgtggtcgagcaggctgaaggagtggagtgcgatttcagtcctctgctgtcagggaccccccctcaggtgtacaacttcaagcggctggtctttactaactgtaactacaatctgaccaagctgctgtcactgttcagcgtgaatgactttacatgctcccagatcagccccgcagccattgctagtaactgttactcctctctgatcctggactacttctcatatccactgagtatgaagagcgacctgagcgtgagttcagccggccccatcagccagttcaactataaacagagcttcagcaatcctacatgcctgattctggctactgtgccacataatctgactaccatcactaagcccctgaaatactcctatattaacaagtgcagccggttcctgtccgacgatagaaccgaagtgccacagctggtcaacgccaatcagtactctccctgtgtgagtatcgtcccttcaaccgtgtgggaagacggggattactatagaaaacagctgagccccctggagggaggaggatggctggtggcatccggatctacagtcgccatgactgagcagctgcagatggggttcggaatcacagtgcagtacggcacagacactaactctgtctgtcccaagctggaattcgctaacgatactaagatcgcaagtcagctgggaaactgcgtggagtactctctgtatggcgtgagtggcagaggggtcttccagaattgtaccgcagtgggcgtccgacagcagcggtttgtgtacgacgcctatcagaatctggtcggctactatagcgacgatgggaactactattgcctgagggcctgtgtgagcgtccctgtgtccgtcatctacgataaggaaaccaaaacacacgccacactgttcgggtccgtggcttgcgagcatattagctccacaatgtctcagtacagtagatcaactaggtcaatgctgaagaggcgcgatagcacctatggacctctgcagacaccagtggggtgtgtcctgggactggtgaactctagtctgtttgtcgaggactgcaagctgcccctgggccagagcctgtgcgccctgcccgacacccccagcaccctgaccccccggagcgtgcggagcgtgcccggcgagatgcggctggccagcatcgccttcaaccaccccatccaggtggaccagctgaacagcagctacttcaagctgagcatccccaccaacttcagcttcggcgtgacccaggagtacatccagaccaccatccagaaggtgaccgtggactgcaagcagtacgtgtgcaacggcttccagaagtgcgagcagctgctgcgggagtacggccagttctgcagcaagatcaaccaggccctgcacggcgccaacctgcggcaggacgacagcgtgcggaacctgttcgccagcgtgaagagcagccagagcagccccatcatccccggcttcggcggcgacttcaacctgaccctgctggagcccgtgagcatcagcaccggcagccggagcgcccggagcgccatcgaggacctgctgttcgacaaggtgaccatcgccgaccccggctacatgcagggctacgacgactgcatgcagcagggccccgccagcgcccgggacctgatctgcgcccagtacgtggccggctacaaggtgctgccccccctgatggacgtgaacatggaggccgcctacaccagcagcctgctgggcagcatcgccggcgtgggctggaccgccggcctgagcagcttcgccgccatccccttcgcccagagcatcttctaccggctgaacggcgtgggcatcacccagcaggtgctgagcgagaaccagaagctgatcgccaacaagttcaaccaggccctgggcgccatgcagaccggcttcaccaccaccaacgaggccttccacaaggtgcaggacgccgtgaacaacaacgcccaggccctgagcaagctggccagcgagctgagcaacaccttcggcgccatcagcgccagcatcggcgacatcatccagcggctggacgtgctggagcaggacgcccagatcgaccggctgatcaacggccggctgaccaccctgaacgccttcgtggcccagcagctggtgcggagcgagagcgccgccctgagcgcccagctggccaaggacaaggtgaacgagtgcgtgaaggcccagagcaagcggagcggcttctgcggccagggcacccacatcgtgagcttcgtggtgaacgcccccaacggcctgtacttcatgcacgtgggctactaccccagcaaccacatcgaggtggtgagcgcctacggcctgtgcgacgccgccaaccccaccaactgcatcgcccccgtgaacggctacttcatcaagaccaacaacacccggatcgtggacgagtggagctacaccggcagcagcttctacgcccccgagcccatcaccagcctgaacaccaagtacgtggccccccaggtgacctaccagaacatcagcaccaacctgcccccccccctgctgggcaacagcaccggcatcgacttccaggacgagctggacgagttcttcaagaacgtgagcaccagcatccccaacttcggcagcctgacccagatcaacaccaccctgctggacctgacctacgagatgctgagcctgcagcaggtggtgaaggccctgaacgagagctacatcgacctgaaggagctgggcaactacacctactacaacaagtggccctggtacatctggctgggcttcatcgccggcctggtggccctggccctgtgcgtgttcttcatcctgtgctgcaccggctgcggcaccaactgcatgggcaagctgaagtgcaaccggtgctgcgaccggtacgaggagtacgacctggagccccacaaggtg cacgtgcactga Anexemplary MERS-CoV S protein, England1 strain is provided as SEQ ID NO:14, below: (SEQ ID NO: 14)MIHSVFLLMFLLTPTESYVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQGLFPYQGDHGDMYVYSAGHATGTTPQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRFFNHTLVLLPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRNASLNSFKEYFNLRNCTFMYTYNITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIPHSIRSIQSDRKAWAAFYVYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSGSVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQLGNCVEYSLYGVSGRGVFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHATLFGSVACEHISSTMSQYSRSTRSMLKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPRSVRSVPGEMRLASIAFNHPIQVDQLNSSYFKLSIPTNFSFGVTQEYIQTTIQKVTVDCKQYVCNGFQKCEQLLREYGQFCSKINQALHGANLRQDDSVRNLFASVKSSQSSPIIPGFGGDFNLTLLEPVSISTGSRSARSAIEDLLFDKVTIADPGYMQGYDDCMQQGPASARDLICAQYVAGYKVLPPLMDVNMEAAYTSSLLGSIAGVGWTAGLSSFAAIPFAQSIFYRLNGVGITQQVLSENQKLIANKFNQALGAMQTGFTTTNEAFHKVQDAVNNNAQALSKLASELSNTFGAISASIGDIIQRLDVLEQDAQIDRLINGRLTTLNAFVAQQLVRSESAALSAQLAKDKVNECVKAQSKRSGFCGQGTHIVSFVVNAPNGLYFMHVGYYPSNHIEVVSAYGLCDAANPTNCIAPVNGYFIKTNNTRIVDEWSYTGSSFYAPEPITSLNTKYVAPQVTYQNISTNLPPPLLGNSTGIDFQDELDEFFKNVSTSIPNFGSLTQINTTLLDLTYEMLSLQQVVKALNESYIDLKELGNYTYYNKWPWYIWLGFIAGLVALALCVFFILCCTGCGTNCMGKLKCNRCCDRYEEYDLEPHKV HVH

Individual precursor S polypeptides form a homotrimer and undergoglycosylation within the Golgi apparatus as well as processing to removethe signal peptide, and cleavage by a cellular protease betweenapproximately position 751/752 to generate separate S1 and S2polypeptide chains, which remain associated as S1/S2 protomers withinthe homotrimer. An exemplary nucleic acid molecule encoding the MERS-CoVS1 protein is provided as SEQ ID NO: 15, below:

MERS-CoV S1 (1-752) encoding sequence, England1 strain: (SEQ ID NO: 15)atgattcactccgtgttcctgctgatgttcctgctgactcctacagagagctatgtggatgtgggacctgattccgtcaagagcgcctgcatcgaagtggacattcagcagaccttctttgataagacatggccaagacccatcgacgtgagcaaagccgatggcatcatctaccctcaggggaggacctattccaatatcacaattacttaccagggcctgttcccatatcagggagaccacggcgatatgtacgtgtattctgctggccatgcaacagggaccacacctcagaagctgtttgtggctaactacagccaggacgtcaaacagttcgcaaatggatttgtggtccgcatcggcgccgctgcaaactctaccggcacagtgatcatttcacctagcacttccgcaaccatccgaaaaatctacccagccttcatgctgggaagctccgtgggcaattttagcgacgggaaaatgggacggttctttaaccacaccctggtgctgctgcctgatggatgcggcacactgctgagggctttctactgtatcctggagccacgcagcggaaaccactgccccgcaggaaatagctacacctcctttgccacatatcatactccagctaccgactgttccgatggcaactacaatcgaaacgcctctctgaatagtttcaaggaatacttcaacctgcggaattgcacattcatgtacacttataacatcaccgaggacgaaattctggagtggttcggaatcactcagaccgcacagggcgtgcacctgttttctagtcgctacgtcgacctgtatggcgggaacatgttccagtttgccactctgcccgtgtacgataccatcaagtactattccatcattcctcattcaatccgcagcattcagtccgatcgaaaggcttgggccgctttctacgtgtataaactgcagccactgaccttcctgctggactttagcgtcgatggctacatccggagagccattgactgcgggtttaatgatctgtcccagctgcactgttcttacgaaagtttcgacgtggagtccggcgtgtattctgtctcaagctttgaggccaagccctctgggagtgtggtcgagcaggctgaaggagtggagtgcgatttcagtcctctgctgtcagggaccccccctcaggtgtacaacttcaagcggctggtctttactaactgtaactacaatctgaccaagctgctgtcactgttcagcgtgaatgactttacatgctcccagatcagccccgcagccattgctagtaactgttactcctctctgatcctggactacttctcatatccactgagtatgaagagcgacctgagcgtgagttcagccggccccatcagccagttcaactataaacagagcttcagcaatcctacatgcctgattctggctactgtgccacataatctgactaccatcactaagcccctgaaatactcctatattaacaagtgcagccggttcctgtccgacgatagaaccgaagtgccacagctggtcaacgccaatcagtactctccctgtgtgagtatcgtcccttcaaccgtgtgggaagacggggattactatagaaaacagctgagccccctggagggaggaggatggctggtggcatccggatctacagtcgccatgactgagcagctgcagatggggttcggaatcacagtgcagtacggcacagacactaactctgtctgtcccaagctggaattcgctaacgatactaagatcgcaagtcagctgggaaactgcgtggagtactctctgtatggcgtgagtggcagaggggtcttccagaattgtaccgcagtgggcgtccgacagcagcggtttgtgtacgacgcctatcagaatctggtcggctactatagcgacgatgggaactactattgcctgagggcctgtgtgagcgtccctgtgtccgtcatctacgataaggaaaccaaaacacacgccacactgttcgggtccgtggcttgcgagcatattagctccacaatgtctcagtacagtagatcaactaggtcaatgctgaagaggcgcgatagcacctatggacctctgcagacaccagtggggtgtgtcctgggactggtgaactctagtctgtttgtcgaggactgcaagctgcccctgggccagagcctgtgcgccctgcccgacacccccagcaccctgaccccccggagcgtg cggagctga Anexemplary MERS-CoV S1 protein sequence, England1 strain, is provided asSEQ ID NO: 16, below (SEQ ID NO: 16)MIHSVFLLMFLLTPTESYVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQGLFPYQGDHGDMYVYSAGHATGTTPQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRFFNHTLVLLPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRNASLNSFKEYFNLRNCTFMYTYNITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIPHSIRSIQSDRKAWAAFYVYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSGSVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQLGNCVEYSLYGVSGRGVFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHATLFGSVACEHISSTMSQYSRSTRSMLKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPRSV RS

The S1 subunit is distal to the virus membrane and contains thereceptor-binding domain (RBD) that mediates virus attachment to its hostreceptor, dipeptidyl peptidase-4 (DPP4). The RBD includes approximatelyresidues 367-606 of the S protein. An exemplary nucleic acid sequenceencoding the MERS-CoV S protein RBD, England1 strain is provided as SEQID NO: 17, below.

(SEQ ID NO: 17) gaggccaagccctctgggagtgtggtcgagcaggctgaaggagtggagtgcgatttcagtcctctgctgtcagggaccccccctcaggtgtacaacttcaagcggctggtctttactaactgtaactacaatctgaccaagctgctgtcactgttcagcgtgaatgactttacatgctcccagatcagccccgcagccattgctagtaactgttactcctctctgatcctggactacttctcatatccactgagtatgaagagcgacctgagcgtgagttcagccggccccatcagccagttcaactataaacagagcttcagcaatcctacatgcctgattctggctactgtgccacataatctgactaccatcactaagcccctgaaatactcctatattaacaagtgcagccggttcctgtccgacgatagaaccgaagtgccacagctggtcaacgccaatcagtactctccctgtgtgagtatcgtcccttcaaccgtgtgggaagacggggattactatagaaaacagctgagccccctggagggaggaggatggctggtggcatccggatctacagtcgccatgactgagcagctgcagatggggttcggaatcacagtgcagtacggcacagacactaactctgtctgtcccaagctggaattcgctaacgatactaagatcgcaagtcagc tgggaaactgcgtggagtacAn exemplary polypeptide sequence of the MERS-CoV S protein RBD isprovided as SEQ ID NO: 18, below: (SEQ ID NO: 18)EAKPSGSVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQLGNCVEY

Another fragment of the MERS-CoV S protein is the ATM fragment, whichincludes the extracellular portion of the S protein. An exemplarynucleic acid sequence encoding the MERS-CoV S ATM fragment is providedas SEQ ID NO: 19, below.

S-ΔTM (VRC 4228) DNA sequence (SEQ ID NO: 19)atgattcactccgtgttcctgctgatgttcctgctgactcctacagagagctatgtggatgtgggacctgattccgtcaagagcgcctgcatcgaagtggacattcagcagaccttctttgataagacatggccaagacccatcgacgtgagcaaagccgatggcatcatctaccctcaggggaggacctattccaatatcacaattacttaccagggcctgttcccatatcagggagaccacggcgatatgtacgtgtattctgctggccatgcaacagggaccacacctcagaagctgtttgtggctaactacagccaggacgtcaaacagttcgcaaatggatttgtggtccgcatcggcgccgctgcaaactctaccggcacagtgatcatttcacctagcacttccgcaaccatccgaaaaatctacccagccttcatgctgggaagctccgtgggcaattttagcgacgggaaaatgggacggttctttaaccacaccctggtgctgctgcctgatggatgcggcacactgctgagggctttctactgtatcctggagccacgcagcggaaaccactgccccgcaggaaatagctacacctcctttgccacatatcatactccagctaccgactgttccgatggcaactacaatcgaaacgcctctctgaatagtttcaaggaatacttcaacctgcggaattgcacattcatgtacacttataacatcaccgaggacgaaattctggagtggttcggaatcactcagaccgcacagggcgtgcacctgttttctagtcgctacgtcgacctgtatggcgggaacatgttccagtttgccactctgcccgtgtacgataccatcaagtactattccatcattcctcattcaatccgcagcattcagtccgatcgaaaggcttgggccgctttctacgtgtataaactgcagccactgaccttcctgctggactttagcgtcgatggctacatccggagagccattgactgcgggtttaatgatctgtcccagctgcactgttcttacgaaagtttcgacgtggagtccggcgtgtattctgtctcaagctttgaggccaagccctctgggagtgtggtcgagcaggctgaaggagtggagtgcgatttcagtcctctgctgtcagggaccccccctcaggtgtacaacttcaagcggctggtctttactaactgtaactacaatctgaccaagctgctgtcactgttcagcgtgaatgactttacatgctcccagatcagccccgcagccattgctagtaactgttactcctctctgatcctggactacttctcatatccactgagtatgaagagcgacctgagcgtgagttcagccggccccatcagccagttcaactataaacagagcttcagcaatcctacatgcctgattctggctactgtgccacataatctgactaccatcactaagcccctgaaatactcctatattaacaagtgcagccggttcctgtccgacgatagaaccgaagtgccacagctggtcaacgccaatcagtactctccctgtgtgagtatcgtcccttcaaccgtgtgggaagacggggattactatagaaaacagctgagccccctggagggaggaggatggctggtggcatccggatctacagtcgccatgactgagcagctgcagatggggttcggaatcacagtgcagtacggcacagacactaactctgtctgtcccaagctggaattcgctaacgatactaagatcgcaagtcagctgggaaactgcgtggagtactctctgtatggcgtgagtggcagaggggtcttccagaattgtaccgcagtgggcgtccgacagcagcggtttgtgtacgacgcctatcagaatctggtcggctactatagcgacgatgggaactactattgcctgagggcctgtgtgagcgtccctgtgtccgtcatctacgataaggaaaccaaaacacacgccacactgttcgggtccgtggcttgcgagcatattagctccacaatgtctcagtacagtagatcaactaggtcaatgctgaagaggcgcgatagcacctatggacctctgcagacaccagtggggtgtgtcctgggactggtgaactctagtctgtttgtcgaggactgcaagctgcccctgggccagagcctgtgcgccctgcccgacacccccagcaccctgaccccccggagcgtgcggagcgtgcccggcgagatgcggctggccagcatcgccttcaaccaccccatccaggtggaccagctgaacagcagctacttcaagctgagcatccccaccaacttcagcttcggcgtgacccaggagtacatccagaccaccatccagaaggtgaccgtggactgcaagcagtacgtgtgcaacggcttccagaagtgcgagcagctgctgcgggagtacggccagttctgcagcaagatcaaccaggccctgcacggcgccaacctgcggcaggacgacagcgtgcggaacctgttcgccagcgtgaagagcagccagagcagccccatcatccccggcttcggcggcgacttcaacctgaccctgctggagcccgtgagcatcagcaccggcagccggagcgcccggagcgccatcgaggacctgctgttcgacaaggtgaccatcgccgaccccggctacatgcagggctacgacgactgcatgcagcagggccccgccagcgcccgggacctgatctgcgcccagtacgtggccggctacaaggtgctgccccccctgatggacgtgaacatggaggccgcctacaccagcagcctgctgggcagcatcgccggcgtgggctggaccgccggcctgagcagcttcgccgccatccccttcgcccagagcatcttctaccggctgaacggcgtgggcatcacccagcaggtgctgagcgagaaccagaagctgatcgccaacaagttcaaccaggccctgggcgccatgcagaccggcttcaccaccaccaacgaggccttccacaaggtgcaggacgccgtgaacaacaacgcccaggccctgagcaagctggccagcgagctgagcaacaccttcggcgccatcagcgccagcatcggcgacatcatccagcggctggacgtgctggagcaggacgcccagatcgaccggctgatcaacggccggctgaccaccctgaacgccttcgtggcccagcagctggtgcggagcgagagcgccgccctgagcgcccagctggccaaggacaaggtgaacgagtgcgtgaaggcccagagcaagcggagcggcttctgcggccagggcacccacatcgtgagcttcgtggtgaacgcccccaacggcctgtacttcatgcacgtgggctactaccccagcaaccacatcgaggtggtgagcgcctacggcctgtgcgacgccgccaaccccaccaactgcatcgcccccgtgaacggctacttcatcaagaccaacaacacccggatcgtggacgagtggagctacaccggcagcagcttctacgcccccgagcccatcaccagcctgaacaccaagtacgtggccccccaggtgacctaccagaacatcagcaccaacctgcccccccccctgctgggcaacagcaccggcatcgacttccaggacgagctggacgagttcttcaagaacgtgagcaccagcatccccaacttcggcagcctgacccagatcaacaccaccctgctggacctgacctacgagatgctgagcctgcagcaggtggtgaaggccctgaacgagagctacatcgacctga aggagctgggcaactacaccAn exemplary polypeptide sequence of the MERS-CoV S ATM fragment isprovided as SEQ ID NO: 20, below. S-ΔTM (VRC 4228) amino acid sequence(SEQ ID NO: 20) MIHSVFLLMFLLTPTESYVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQGLFPYQGDHGDMYVYSAGHATGTTPQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRFFNHTLVLLPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRNASLNSFKEYFNLRNCTFMYTYNITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIPHSIRSIQSDRKAWAAFYVYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSGSVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQLGNCVEYSLYGVSGRGVFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHATLFGSVACEHISSTMSQYSRSTRSMLKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPRSVRSVPGEMRLASIAFNHPIQVDQLNSSYFKLSIPTNFSFGVTQEYIQTTIQKVTVDCKQYVCNGFQKCEQLLREYGQFCSKINQALHGANLRQDDSVRNLFASVKSSQSSPIIPGFGGDFNLTLLEPVSISTGSRSARSAIEDLLFDKVTIADPGYMQGYDDCMQQGPASARDLICAQYVAGYKVLPPLMDVNMEAAYTSSLLGSIAGVGWTAGLSSFAAIPFAQSIFYRLNGVGITQQVLSENQKLIANKFNQALGAMQTGFTTTNEAFHKVQDAVNNNAQALSKLASELSNTFGAISASIGDIIQRLDVLEQDAQIDRLINGRLTTLNAFVAQQLVRSESAALSAQLAKDKVNECVKAQSKRSGFCGQGTHIVSFVVNAPNGLYFMHVGYYPSNHIEVVSAYGLCDAANPTNCIAPVNGYFIKTNNTRIVDEWSYTGSSFYAPEPITSLNTKYVAPQVTYQNISTNLPPPLLGNSTGIDFQDELDEFFKNVSTSIPNFGSLTQINTTLLDLTYEMLSLQQVVKALNESYIDLKELGNYT

The MERS-CoV S protein or fragments thereof can be isolated from nativesources or produced using recombinant techniques, or chemically orenzymatically synthesized.

Analogs and variants of the MERS-CoV S protein or fragments thereof maybe used in the methods and systems of the present invention. Through theuse of recombinant DNA technology, variants of the MERS-CoV S protein orfragments thereof may be prepared by altering the underlying DNA. Allsuch variations or alterations in the structure of the MERS-CoV Sprotein or fragments thereof resulting in variants are included withinthe scope of this invention. Such variants include insertions,substitutions, or deletions of one or more amino acid residues,glycosylation variants, unglycosylated The MERS-CoV S protein orfragments thereof, organic and inorganic salts, covalently modifiedderivatives of the MERS-CoV S protein or fragments thereof, or aprecursor thereof. Such variants may maintain one or more of thefunctional, biological activities of the MERS-CoV S protein or fragmentthereof, such as binding to DPP4. The MERS-CoV S protein or a fragmentthereof (e.g., a MERS-CoV S1 protein) can be modified, for example, byPEGylation, to increase the half-life of the protein in the recipient,to retard clearance from the pericardial space, and/or to make theprotein more stable for delivery to a subject.

In some embodiments, a MERS-CoV S protein or fragment thereof usefulwithin the disclosure is modified to produce peptide mimetics byreplacement of one or more naturally occurring side chains of the 20genetically encoded amino acids (or D-amino acids) with other sidechains, for example with groups such as alkyl, lower alkyl, cyclic 4-,5-, 6-, to 7-membered alkyl, amide, amide lower alkyl, amide di(loweralkyl), lower alkoxy, hydroxy, carboxy and the lower ester derivativesthereof, and with 4-, 5-, 6-, to 7-membered heterocyclics. For example,proline analogs can be made in which the ring size of the prolineresidue is changed from a 5-membered ring to a 4, 6-, or 7-memberedring. Cyclic groups can be saturated or unsaturated, and if unsaturated,can be aromatic or non-aromatic. Heterocyclic groups can contain one ormore nitrogen, oxygen, and/or sulphur heteroatoms. Examples of suchgroups include furazanyl, furyl, imidazolidinyl, imidazolyl,imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (e.g., morpholino),oxazolyl, piperazinyl (e.g., 1-piperazinyl), piperidyl (e.g.,1-piperidyl, piperidino), pyranyl, pyrazinyl, pyrazolidinyl,pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl(e.g., 1-pyrrolidinyl), pyrrolinyl, pyrrolyl, thiadiazolyl, thiazolyl,thienyl, thiomorpholinyl (e.g., thiomorpholino), and triazolyl groups.These heterocyclic groups can be substituted or unsubstituted. Where agroup is substituted, the substituent can be alkyl, alkoxy, halogen,oxygen, or substituted or unsubstituted phenyl. Peptides, as well aspeptide analogs and mimetics, can also be covalently bound to one ormore of a variety of nonproteinaceous polymers, for example,polyethylene glycol, polypropylene glycol, or polyoxyalkenes, asdescribed in U.S. Pat. Nos. 4,640,835; 4,496,668; 4,301,144; 4,668,417;4,791,192; and 4,179,337.

In addition to the naturally occurring genetically encoded amino acids,amino acid residues in a MERS-CoV S protein or fragment thereof may besubstituted with naturally occurring non-encoded amino acids andsynthetic amino acids. Certain commonly encountered amino acids whichprovide useful substitutions include, but are not limited to, β-alanineand other omega-amino acids, such as 3-aminopropionic acid,2,3-diaminopropionic acid, 4-aminobutyric acid and the like;α-aminoisobutyric acid; ε-aminohexanoic acid; δ-aminovaleric acid;N-methylglycine or sarcosine; ornithine; citrulline; t-butylalanine;t-butylglycine; N-methylisoleucine; phenylglycine; cyclohexylalanine;norleucine; naphthylalanine; 4-chlorophenylalanine;2-fluorophenylalanine; 3-fluorophenylalanine; 4-fluorophenylalanine;penicillamine; 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid;β-2-thienylalanine; methionine sulfoxide; homoarginine; N-acetyl lysine;2,4-diaminobutyric acid; 2,3-diaminobutyric acid; p-aminophenylalanine;N-methyl valine; homocysteine; homophenylalanine; homoserine;hydroxyproline; homoproline; N-methylated amino acids; and peptoids(N-substituted glycines).

While in certain embodiments, the amino acids of a MERS-CoV S protein orfragment thereof will be substituted with L-amino acids; however, thesubstitutions are not limited to L-amino acids. Thus, also encompassedby the present disclosure are modified forms of the SAHPs, wherein anL-amino acid is replaced with an identical D-amino acid (e.g.,L-Arg→D-Arg) or with a conservatively-substituted D-amino acid (e.g.,L-Arg→D-Lys), and vice versa.

Other peptide analogs and mimetics within the scope of the disclosureinclude glycosylation variants, and covalent or aggregate conjugateswith other chemical moieties. Covalent derivatives can be prepared bylinkage of functionalities to groups which are found in amino acid sidechains or at the N- or C-termini, by means which are well known in theart. These derivatives can include, without limitation, aliphatic estersor amides of the carboxyl terminus, or of residues containing carboxylside chains, O-acyl derivatives of hydroxyl group-containing residues,and N-acyl derivatives of the amino terminal amino acid or amino-groupcontaining residues (e.g., lysine or arginine). Acyl groups are selectedfrom the group of alkyl-moieties including C3 to C18 normal alkyl,thereby forming alkanoyl aroyl species. Also embraced are versions of anative primary amino acid sequence which have other minor modifications,including phosphorylated amino acid residues, for example,phosphotyrosine, phosphoserine, or phosphothreonine, or other moieties,including ribosyl groups or cross-linking reagents.

In another embodiment, an additional functional domain or peptide can belinked to a MERS-CoV S protein or fragment or analog disclosed herein,creating a peptide/peptide analog-additional functional domain/peptideconjugate. The additional functional domain or peptide can be linked tothe RLX polypeptide or peptide analog at either the N- and/orC-terminus.

Optionally, a linker can be included between the MERS-CoV S protein orfragment or analog and the additional functional domain or peptide. Thelinkers contemplated by the present disclosure can be any bifunctionalmolecule capable of covalently linking two peptides to one another.Thus, suitable linkers are bifunctional molecules in which thefunctional groups are capable of being covalently attached to the N-and/or C-terminus of a peptide. Functional groups suitable forattachment to the N- or C-terminus of peptides are well known in theart, as are suitable chemistries for effecting such covalent bondformation. The linker may be flexible, rigid or semi-rigid. Suitablelinkers include, for example, amino acid residues such as Pro or Gly orpeptide segments containing from about 2 to about 5, 10, 15, 20, or evenmore amino acids, bifunctional organic compounds such asH₂N(CH₂)_(n)COOH where n is an integer from 1 to 12, and the like.Examples of such linkers, as well as methods of making such linkers andpeptides incorporating such linkers, are well-known in the art (see,e.g., Hunig et al., Chem. Ber. 100:3039-3044, 1974 and Basak et al.,Bioconjug. Chem. 5:301-305, 1994).

Conjugation methods applicable to the present disclosure include, by wayof non-limiting example, reductive amination, diazo coupling, thioetherbond, disulfide bond, amidation and thiocarbamoyl chemistries. In oneembodiment, the amphipathic alpha-helical domains are “activated” priorto conjugation. Activation provides the necessary chemical groups forthe conjugation reaction to occur. In one specific, non-limitingexample, the activation step includes derivatization with adipic aciddihydrazide. In another specific, non-limiting example, the activationstep includes derivatization with the N-hydroxysuccinimide ester of3-(2-pyridyl dithio)-propionic acid. In yet another specific,non-limiting example, the activation step includes derivatization withsuccinimidyl 3-(bromoacetamido) propionate. Further, non-limitingexamples of derivatizing agents include succinimidylformylbenzoate andsuccinimidyllevulinate.

Also encompassed by the present disclosure are polypeptides includingdimers, trimers, tetramers and even higher order polymers (i.e.,“multimers”) comprising the same or different MERS-CoV S protein orfragment thereof. In multimers, the MERS-CoV S protein or fragmentthereof may be directly attached to one another or separated by one ormore linkers. The MERS-CoV S protein or fragment thereof can beconnected in a head-to-tail fashion (i.e., N-terminus to C-terminus), ahead-to-head fashion, (i.e., N-terminus to N-terminus), a tail-to-tailfashion (i.e., C-terminus to C-terminus), and/or combinations thereof.In one embodiment, the multimers are tandem repeats of two, three, four,and up to about ten MERS-CoV S proteins or fragments thereof, but anynumber of MERS-CoV S proteins or fragments thereof can be used.

B. Protein Nanoparticles

In some embodiments a protein nanoparticle is provided that includes oneor more of the disclosed MERS-CoV S protein or fragment thereof (e.g., aMERS-CoV S protein or S1 protein, or RBD). Non-limiting example ofnanoparticles include ferritin nanoparticles, encapsulin nanoparticles,Sulfur Oxygenase Reductase (SOR) nanoparticles, and lumazine synthasenanoparticles, which are comprised of an assembly of monomeric subunitsincluding ferritin proteins, encapsulin proteins, SOR proteins, andlumazine synthase, respectively. Exemplary sequences of recombinantMERS-CoV S protein or fragment thereof linked to a nanoparticle subunitare provided below. To construct protein nanoparticles including aMERS-CoV S protein or immunogenic fragment thereof, the MERS-CoV Sprotein or fragment can be linked to a subunit of the proteinnanoparticle (such as a ferritin protein, an encapsulin protein, a SORprotein, or a lumazine synthase protein). The fusion proteinself-assembles into a nanoparticle under appropriate conditions.

In several embodiments, the protein nanoparticle comprises two or moreMERS-CoV S proteins or immunogenic fragments thereof, wherein the two ormore MERS-CoV S proteins or fragments are from at least two differentstrains of MERS-CoV.

In some embodiments, a MERS-CoV S protein or immunogenic fragmentthereof can be linked to a ferritin subunit to construct a ferritinnanoparticle. Ferritin nanoparticles and their use for immunizationpurposes (e.g., for immunization against influenza antigens) have beendisclosed in the art (see, e.g., Kanekiyo et al., Nature, 499:102-106,2013, incorporated by reference herein in its entirety). Ferritin is aglobular protein that is found in all animals, bacteria, and plants, andwhich acts primarily to control the rate and location of polynuclearFe(III)₂O₃ formation through the transportation of hydrated iron ionsand protons to and from a mineralized core. The globular form of theferritin nanoparticle is made up of monomeric subunits, which arepolypeptides having a molecule weight of approximately 17-20 kDa. Anexample of the amino acid sequence of one such monomeric ferritinsubunit is represented by:

(SEQ ID NO: 21) ESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS

Each monomeric subunit has the topology of a helix bundle which includesa four antiparallel helix motif, with a fifth shorter helix (thec-terminal helix) lying roughly perpendicular to the long axis of the 4helix bundle. According to convention, the helices are labeled ‘A, B, C,D & E’ from the N-terminus respectively. The N-terminal sequence liesadjacent to the capsid three-fold axis and extends to the surface, whilethe E helices pack together at the four-fold axis with the C-terminusextending into the capsid core. The consequence of this packing createstwo pores on the capsid surface. It is expected that one or both ofthese pores represent the point by which the hydrated iron diffuses intoand out of the capsid. Following production, these monomeric subunitproteins self-assemble into the globular ferritin protein. Thus, theglobular form of ferritin comprises 24 monomeric, subunit proteins, andhas a capsid-like structure having 432 symmetry. Methods of constructingferritin nanoparticles are known to the person of ordinary skill in theart and are further described herein (see, e.g., Zhang, Int. J. Mol.Sci., 12:5406-5421, 2011, which is incorporated herein by reference inits entirety).

In specific examples, the ferritin polypeptide is E. coli ferritin,Helicobacter pylori ferritin, human light chain ferritin, bullfrogferritin or a hybrid thereof, such as E. coli-human hybrid ferritin, E.coli-bullfrog hybrid ferritin, or human-bullfrog hybrid ferritin.Exemplary amino acid sequences of ferritin polypeptides and nucleic acidsequences encoding ferritin polypeptides for use to make a ferritinnanoparticle including a MERS-CoV S protein or immunogenic fragmentthereof can be found in GENBANK®, for example at accession numbersZP_03085328, ZP_06990637, EJB64322.1, AAA35832, NP_000137 AAA49532,AAA49525, AAA49524 and AAA49523, which are specifically incorporated byreference herein in their entirety as available Jun. 20, 2014. In someembodiments, a MERS-CoV S protein or fragment thereof can be linked to aferritin subunit including an amino acid sequence at least 80% (such asat least 85%, at least 90%, at least 95%, or at least 97%) identical toamino acid sequence set forth as SEQ ID NO: 21.

In some embodiments, the RBD domain of MERS-CoV S protein can be linkedto a ferritin nanoparticle subunit. For example, the RBD domain cancomprise or consist of amino acids 367-601, 367-606, or 381-588 of theMERS-CoV S protein sequence set forth as SEQ ID NO: 14, and can belinked to a ferritin nanoparticle subunit. Specific examples ofpolypeptide sequences of the RBD domain of MERS-CoV S protein linked toa ferritin nanoparticle subunit are provided as the amino acid sequencesset forth as SEQ ID NOs: 22 and 23, below. In some embodiments, the RBDdomain linked to the protein nanoparticle subunit includes an amino acidsequence at least 80% (such as at least 90%, at least 95%, or 100%)identical to the sequence set forth as one of SEQ ID NOs: 22 or 23.

MERS-CoV S-RBD(367-601)_506F_ferritin (SEQ ID NO: 22)EAKPSGSVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQLGSGESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQY VKGIAKSRKSGS MERS-CoVS-RBD(381-588)_506F_ferritin (SEQ ID NO: 23)VECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLSGESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKSGSFor production purposes, the RBD domain linked to the nanoparticlesubunit can include a signal peptide that is cleaved during cellularprocessing. For example, the RBD domain linked to the proteinnanoparticle can include a signal peptide at its N-terminus includingthe amino acid sequence set forth as:

bPRL(LA) signal peptide (SEQ ID NO: 24) MDSKGSSQKGSRLLLLLVVSNLLLPQGVLAhCD5 signal peptide (SEQ ID NO: 25) MPMGSLQPLATLYLLGMLVASVLAExemplary nucleic acid molecule sequences encoding the disclosed RBDdomains linked to a ferritin nanoparticle sequence are provided as SEQID NOs: 26 and 27, below.

CMV8xR-bPRL(LA)-MERS-CoV S-RBD(367-601)_506F_ferritin (SEQ ID NO: 26)

CMV8xR-bPRL(LA)-MERS-CoV S-RBD(381-588)_506F_ferritin (SEQ ID NO: 27)

In additional embodiments, any of the disclosed MERS-CoV S proteins orfragments thereof can be linked to a lumazine synthase subunit toconstruct a lumazine synthase nanoparticle. The globular form oflumazine synthase nanoparticle is made up of monomeric subunits; anexample of the sequence of one such lumazine synthase subunit isprovides as the amino acid sequence set forth as:

(SEQ ID NO: 28) MQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLF KSLR.

In some embodiments, a disclosed MERS-CoV S protein or immunogenicfragment thereof can be linked to a lumazine synthase subunit includingan amino acid sequence at least 80% (such as at least 85%, at least 90%,at least 95%, or at least 97%) identical to amino acid sequence setforth as SEQ ID NO: 28.

In additional embodiments, the MERS-CoV S protein or immunogenicfragment thereof can be linked to an encapsulin nanoparticle subunit toconstruct an encapsulin nanoparticle. The globular form of theencapsulin nanoparticle is made up of monomeric subunits; an example ofthe sequence of one such encapsulin subunit is provides as the aminoacid sequence set forth as

(SEQ ID NO: 29) MEFLKRSFAPLTEKQWQEIDNRAREIFKTQLYGRKFVDVEGPYGWEYAAHPLGEVEVLSDENEVVKWGLRKSLPLIELRATFTLDLWELDNLERGKPNVDLSSLEETVRKVAEFEDEVIFRGCEKSGVKGLLSFEERKIECGSTPKDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINFLKEEAGHYPLEKRVEECLRGGKIITTPRIEDALVVSERGGDFKLILGQDLSIGYEDREKDAVRLFITETF TFQVVNPEALILLKF.

In some embodiments, a MERS-CoV S protein or immunogenic fragmentthereof can be linked to an encapsulin subunit including an amino acidsequence at least 80% (such as at least 85%, at least 90%, at least 95%,or at least 97%) identical to amino acid sequence set forth as SEQ IDNO: 29.

Encapsulin proteins are a conserved family of bacterial proteins alsoknown as linocin-like proteins that form large protein assemblies thatfunction as a minimal compartment to package enzymes. The encapsulinassembly is made up of monomeric subunits, which are polypeptides havinga molecule weight of approximately 30 kDa. Following production, themonomeric subunits self-assemble into the globular encapsulin assemblyincluding 60, or in some cases, 180 monomeric subunits. Methods ofconstructing encapsulin nanoparticles are known to the person ofordinary skill in the art, and further described herein (see, forexample, Sutter et al., Nature Struct. and Mol. Biol., 15:939-947, 2008,which is incorporated by reference herein in its entirety). In specificexamples, the encapsulin polypeptide is bacterial encapsulin, such asThermotoga maritime or Pyrococcus furiosus or Rhodococcus erythropolisor Myxococcus xanthus encapsulin.

In some embodiments, the RBD domain of MERS-CoV S protein can be linkedto an encapsulin nanoparticle subunit. For example, the RBD domain cancomprise or consist of amino acids 367-601, 367-606, or 381-588 of theMERS-CoV S protein sequence set forth as SEQ ID NO: 14 and can be linkedto an encapsulin nanoparticle subunit. Specific examples of polypeptidesequences of the RBD domain of MERS-CoV S protein linked to anencapsulin nanoparticle subunit are provided as the amino acid sequencesset forth as SEQ ID NO: 30 and 31, below. In some embodiments, the RBDdomain linked to the encapsulin nanoparticle subunit includes an aminoacid sequence at least 80% (such as at least 90%, at least 95%, or 100%)identical to the sequence set forth as one of SEQ ID NOs: 30 or 31. Forproduction purposes, the RBD domain linked to the encapsulinnanoparticle subunit can include a signal peptide that is cleaved duringcellular processing. For example, the RBD domain linked to the proteinnanoparticle can include a signal peptide at its N-terminus includingthe amino acid sequence set forth as SEQ ID NO: 24 or SEQ ID NO: 25.

MERS-CoV S-RBD(367-601)_506F_encapsulin (SEQ ID NO: 30)MEFLKRSFAPLTEKQWQEIDNRAREIFKTQLYGRKFVDVEGPYGWEYAAHPLGEVEVLSDENEVVKWGLRKSLPLIELRATFTLDLWELDNLERGKPNVDLSSLEETVRKVAEFEDEVIFRGCEKSGVKGLLSFEERKIECGSTPKDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINFLKEEAGHYPLEKRVEECLRGGKIITTPRIEDALVVSERGGDFKLILGQDLSIGYEDREKDAVRLFITETFTFQVVNPEALILLKSGEAKPSGSVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQL G MERS-CoVS-RBD(381-588)_506F_encapsulin (SEQ ID NO: 31)MEFLKRSFAPLTEKQWQEIDNRAREIFKTQLYGRKFVDVEGPYGWEYAAHPLGEVEVLSDENEVVKWGLRKSLPLIELRATFTLDLWELDNLERGKPNVDLSSLEETVRKVAEFEDEVIFRGCEKSGVKGLLSFEERKIECGSTPKDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINFLKEEAGHYPLEKRVEECLRGGKIITTPRIEDALVVSERGGDFKLILGQDLSIGYEDREKDAVRLFITETFTFQVVNPEALILLKSGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLExemplary nucleic acid molecule sequences encoding the disclosed RBDdomains linked to an encapsulin nanoparticle subunit are provided as SEQID NOs: 32 and 33.

CMV8xR-hCD5-MERS-CoV S-RBD(367-601)_506F_encapsulin (SEQ ID NO: 32)CMV8xR-hCD5-MERS-CoV S-RBD(381-588)_506F_encapsulin (SEQ ID NO: 33)

In additional embodiments, a MERS-CoV S protein or immunogenic fragmentthereof can be linked to a Sulfur Oxygenase Reductase (SOR) subunit toconstruct a recombinant SOR nanoparticle. In some embodiments, the SORsubunit can include the amino acid sequence set forth as

(SEQ ID NO: 34) MEFLKRSFAPLTEKQWQEIDNRAREIFKTQLYGRKFVDVEGPYGWEYAAHPLGEVEVLSDENEVVKWGLRKSLPLIELRATFTLDLWELDNLERGKPNVDLSSLEETVRKVAEFEDEVIFRGCEKSGVKGLLSFEERKIECGSTPKDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINFLKEEAGHYPLEKRVEECLRGGKIITTPRIEDALVVSERGGDFKLILGQDLSIGYEDREKDAVRLFITETF TFQVVNPEALILLKF.

In some embodiments, a MERS-CoV S protein or immunogenic fragmentthereof can be linked to a SOR subunit including an amino acid sequenceat least 80% (such as at least 85%, at least 90%, at least 95%, or atleast 97%) identical to amino acid sequence set forth as SEQ ID NO: 34.

SOR proteins are microbial proteins (for example from thethermoacidophilic archaeon Acidianus ambivalens that form 24 subunitprotein assemblies. Methods of constructing SOR nanoparticles are knownto the person of ordinary skill in the art (see, e.g., Urich et al.,Science, 311:996-1000, 2006, which is incorporated by reference hereinin its entirety). An example of an amino acid sequence of a SOR proteinfor use to make SOR nanoparticles is set forth in Urich et al., Science,311:996-1000, 2006, which is incorporated by reference herein in itsentirety.

In some examples, the MERS-CoV S protein or immunogenic fragment thereofcan be linked to the N- or C-terminus, or placed within an internal loopof a ferritin, encapsulin, SOR, or lumazine synthase subunit, forexample with a linker, such as a Ser-Gly linker. When the constructshave been made in HEK 293 Freestyle cells, the fusion proteins aresecreted from the cells and self-assembled into nanoparticles. Thenanoparticles can be purified using known techniques, for example by afew different chromatography procedures, e.g. Mono Q (anion exchange)followed by size exclusion (SUPEROSE® 6) chromatography.

Several embodiments include a monomeric subunit of a ferritin,encapsulin, SOR, or lumazine synthase protein, or any portion thereofwhich is capable of directing self-assembly of monomeric subunits intothe globular form of the protein. Amino acid sequences from monomericsubunits of any known ferritin, encapsulin, SOR, or lumazine synthaseprotein can be used to produce fusion proteins with the MERS-CoV Sprotein or immunogenic fragment thereof, so long as the monomericsubunit is capable of self-assembling into a nanoparticle displaying theMERS-CoV S protein or immunogenic fragment thereof on its surface.

The fusion proteins need not comprise the full-length sequence of amonomeric subunit polypeptide of a ferritin, encapsulin, SOR, orlumazine synthase protein. Portions, or regions, of the monomericsubunit polypeptide can be utilized so long as the portion comprisesamino acid sequences that direct self-assembly of monomeric subunitsinto the globular form of the protein.

In some embodiments, it may be useful to engineer mutations into theamino acid sequence of the monomeric ferritin, encapsulin, SOR, orlumazine synthase subunits. For example, it may be useful to alter sitessuch as enzyme recognition sites or glycosylation sites in order to givethe fusion protein beneficial properties (e.g., half-life).

It will be understood by those skilled in the art that fusion of any ofthe MERS-CoV S protein (e.g., in trimeric form) or immunogenic fragmentthereof (such as RBD domain) to the ferritin, encapsulin, SOR, orlumazine synthase protein should be done such that the MERS-CoV Sprotein or immunogenic fragment thereof does not interfere withself-assembly of the monomeric ferritin, encapsulin, SOR, or lumazinesynthase subunits into the globular protein, and that the ferritin,encapsulin, SOR, or lumazine synthase subunits do not interfere with theability of the disc MERS-CoV S protein or immunogenic fragment thereofto elicit an immune response to MERS-CoV. In some embodiments, theferritin, encapsulin, SOR, or lumazine synthase protein and MERS-CoV Sprotein or immunogenic fragment thereof can be joined together directlywithout affecting the activity of either portion. In other embodiments,the ferritin, encapsulin, SOR, or lumazine synthase protein and theMERS-CoV S protein or immunogenic fragment thereof can be joined using alinker (also referred to as a spacer) sequence. The linker sequence isdesigned to position the ferritin, encapsulin, SOR, or lumazine synthaseportion of the fusion protein and the MERS-CoV S protein or immunogenicfragment thereof can be linked to an portion of the fusion protein, withregard to one another, such that the fusion protein maintains theability to assemble into nanoparticles, and also elicit an immuneresponse to MERS-CoV. In several embodiments, the linker sequencescomprise amino acids. Preferable amino acids to use are those havingsmall side chains and/or those which are not charged. Such amino acidsare less likely to interfere with proper folding and activity of thefusion protein. Accordingly, preferred amino acids to use in linkersequences, either alone or in combination are serine, glycine andalanine. One example of such a linker sequence is SGG. Amino acids canbe added or subtracted as needed. Those skilled in the art are capableof determining appropriate linker sequences for construction of proteinnanoparticles.

C. Virus-Like Particles

In some embodiments, a virus-like particle (VLP) is provided thatincludes a disclosed immunogen (e.g., a MERS-CoV S protein orimmunogenic fragment thereof). VLPs lack the viral components that arerequired for virus replication and thus represent a highly attenuatedform of a virus. The VLP can display a polypeptide (e.g., a MERS-CoV Sprotein or immunogenic fragment thereof) that is capable of eliciting animmune response to MERS-CoV when administered to a subject. Virus likeparticles and methods of their production are known and familiar to theperson of ordinary skill in the art, and viral proteins from severalviruses are known to form VLPs, including human papillomavirus, HIV(Kang et al., Biol. Chem. 380: 353-64 (1999)), Semliki-Forest virus(Notka et al., Biol. Chem. 380: 341-52 (1999)), Chikungunya virus(Akahata et al., Nat. Med. 16:334-338 (2010)), human polyomavirus(Goldmann et al., J. Virol. 73: 4465-9 (1999)), rotavirus (Jiang et al.,Vaccine 17: 1005-13 (1999)), parvovirus (Casal, Biotechnology andApplied Biochemistry, Vol 29, Part 2, pp 141-150 (1999)), canineparvovirus (Hurtado et al., J. Virol. 70: 5422-9 (1996)), hepatitis Evirus (Li et al., J. Virol. 71: 7207-13 (1997)), and Newcastle diseasevirus. The formation of such VLPs can be detected by any suitabletechnique. Examples of suitable techniques known in the art fordetection of VLPs in a medium include, e.g., electron microscopytechniques, dynamic light scattering (DLS), selective chromatographicseparation (e.g., ion exchange, hydrophobic interaction, and/or sizeexclusion chromatographic separation of the VLPs) and density gradientcentrifugation.

D. Viral Vectors

The nucleic acid molecules encoding the disclosed immunogens (e.g.,MERS-CoV S protein or fragment thereof) can be included in a viralvector, for example for expression of the antigen in a host cell, or forimmunization of a subject as disclosed herein. In some embodiments, theviral vectors are administered to a subject as part of a prime-boostvaccination. In several embodiments, the viral vectors are included in avaccine, such as a primer vaccine or a booster vaccine for use in aprime-boost vaccination.

In several examples, the viral vector can be replication-competent. Forexample, the viral vector can have a mutation in the viral genome thatdoes not inhibit viral replication in host cells. The viral vector alsocan be conditionally replication-competent. In other examples, the viralvector is replication-deficient in host cells.

A number of viral vectors have been constructed, that can be used toexpress the disclosed antigens, including polyoma, i.e., SV40 (Madzak etal., 1992, J. Gen. Virol., 73:15331536), adenovirus (Berkner, 1992, Cur.Top. Microbiol. Immunol., 158:39-6; Berliner et al., 1988, BioTechniques, 6:616-629; Gorziglia et al., 1992, J. Virol., 66:4407-4412;Quantin et al., 1992, Proc. Natl. Acad. Sci. USA, 89:2581-2584;Rosenfeld et al., 1992, Cell, 68:143-155; Wilkinson et al., 1992, Nucl.Acids Res., 20:2233-2239; Stratford-Perricaudet et al., 1990, Hum. GeneTher., 1:241-256), vaccinia virus (Mackett et al., 1992, Biotechnology,24:495-499), adeno-associated virus (Muzyczka, 1992, Curr. Top.Microbiol. Immunol., 158:91-123; On et al., 1990, Gene, 89:279-282),herpes viruses including HSV and EBV (Margolskee, 1992, Curr. Top.Microbiol. Immunol., 158:67-90; Johnson et al., 1992, J. Virol.,66:29522965; Fink et al., 1992, Hum. Gene Ther. 3:11-19; Breakfield etal., 1987, Mol. Neurobiol., 1:337-371; Fresse et al., 1990, Biochem.Pharmacol., 40:2189-2199), Sindbis viruses (H. Herweijer et al., 1995,Human Gene Therapy 6:1161-1167; U.S. Pat. Nos. 5,091,309 and5,2217,879), alphaviruses (S. Schlesinger, 1993, Trends Biotechnol.11:18-22; I. Frolov et al., 1996, Proc. Natl. Acad. Sci. USA93:11371-11377) and retroviruses of avian (Brandyopadhyay et al., 1984,Mol. Cell Biol., 4:749-754; Petropouplos et al., 1992, J. Virol.,66:3391-3397), murine (Miller, 1992, Curr. Top. Microbiol. Immunol.,158:1-24; Miller et al., 1985, Mol. Cell Biol., 5:431-437; Sorge et al.,1984, Mol. Cell Biol., 4:1730-1737; Mann et al., 1985, J. Virol.,54:401-407), and human origin (Page et al., 1990, J. Virol.,64:5370-5276; Buchschalcher et al., 1992, J. Virol., 66:2731-2739).Baculovirus (Autographa californica multinuclear polyhedrosis virus;AcMNPV) vectors are also known in the art, and may be obtained fromcommercial sources (such as PharMingen, San Diego, Calif.; ProteinSciences Corp., Meriden, Conn.; Stratagene, La Jolla, Calif.).

In several embodiments, the viral vector can include an adenoviralvector that expresses a disclosed recombinant MERS-CoV S protein orfragment thereof. Adenovirus from various origins, subtypes, or mixtureof subtypes can be used as the source of the viral genome for theadenoviral vector. Non-human adenovirus (e.g., simian, chimpanzee,gorilla, avian, canine, ovine, or bovine adenoviruses) can be used togenerate the adenoviral vector. For example, a simian adenovirus can beused as the source of the viral genome of the adenoviral vector. Asimian adenovirus can be of serotype 1, 3, 7, 11, 16, 18, 19, 20, 27,33, 38, 39, 48, 49, 50, or any other simian adenoviral serotype. Asimian adenovirus can be referred to by using any suitable abbreviationknown in the art, such as, for example, SV, SAdV, SAV or sAV. Agorilla-derived adenovirus vector that is similar to ChAd3 or human Ad5and is a subtype C adenovirus can also be used (see, e.g., Johnson etal., Mol Ther. 2014 January; 22(1):196-205). In some examples, a simianadenoviral vector is a simian adenoviral vector of serotype 3, 7, 11,16, 18, 19, 20, 27, 33, 38, or 39. In one example, a chimpanzee serotypeC Ad3 vector is used (see, e.g., Peruzzi et al., Vaccine, 27:1293-1300,2009). Human adenovirus can be used as the source of the viral genomefor the adenoviral vector. Human adenovirus can be of various subgroupsor serotypes. For instance, an adenovirus can be of subgroup A (e.g.,serotypes 12, 18, and 31), subgroup B (e.g., serotypes 3, 7, 11, 14, 16,21, 34, 35, and 50), subgroup C (e.g., serotypes 1, 2, 5, and 6),subgroup D (e.g., serotypes 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24,25, 26, 27, 28, 29, 30, 32, 33, 36-39, and 42-48), subgroup E (e.g.,serotype 4), subgroup F (e.g., serotypes 40 and 41), an unclassifiedserogroup (e.g., serotypes 49 and 51), or any other adenoviral serotype.The person of ordinary skill in the art is familiar with replicationcompetent and deficient adenoviral vectors (including singly andmultiply replication deficient adenoviral vectors). Examples ofreplication-deficient adenoviral vectors, including multiplyreplication-deficient adenoviral vectors, are disclosed in U.S. Pat.Nos. 5,837,511; 5,851,806; 5,994,106; 6,127,175; 6,482,616; and7,195,896, and International Patent Application Nos. WO 94/28152, WO95/02697, WO 95/16772, WO 95/34671, WO 96/22378, WO 97/12986, WO97/21826, and WO 03/022311.

E. Methods of Inducing an Immune Response

The disclosed immunogens (e.g., MERS-CoV S protein or immunogenicfragment thereof, polynucleotides encoding same, protein nanoparticles,viral-like particles, or vectors) and compositions, can be used inmethods of inducing an immune response to MERS-CoV S protein. In severalembodiments, a therapeutically effective amount of an immunogeniccomposition including one or more of the disclosed immunogens, can beadministered to a subject in order to generate an immune response toMERS-CoV.

In some embodiments, a subject is selected for treatment that has, or isat risk for developing, an MERS-CoV infection, for example because ofexposure or the possibility of exposure to MERS-CoV. Followingadministration of a therapeutically effective amount of a disclosedimmunogen, the subject can be monitored for MERS-CoV infection, symptomsassociated with MERS-CoV infection, or both.

Typical subjects intended for treatment with the therapeutics andmethods of the present disclosure include humans, as well as non-humanprimates and other animals (such as camels). To identify subjects forprophylaxis or treatment according to the methods of the disclosure,accepted screening methods are employed to determine risk factorsassociated with a targeted or suspected disease or condition, or todetermine the status of an existing disease or condition in a subject.These screening methods include, for example, conventional work-ups todetermine environmental, familial, occupational, and other such riskfactors that may be associated with the targeted or suspected disease orcondition, as well as diagnostic methods, such as various ELISA andother immunoassay methods, which are available and well known in the artto detect and/or characterize MERS-CoV infection. These and otherroutine methods allow the clinician to select patients in need oftherapy using the methods and pharmaceutical compositions of thedisclosure. In accordance with these methods and principles, acomposition can be administered according to the teachings herein, orother conventional methods known to the person of ordinary skill in theart, as an independent prophylaxis or treatment program, or as afollow-up, adjunct or coordinate treatment regimen to other treatments.

The administration of a disclosed immunogen (e.g., MERS-CoV S protein orimmunogenic fragment thereof, polynucleotides encoding same, proteinnanoparticles, viral-like particles, or vectors) can be for prophylacticor therapeutic purpose. When provided prophylactically, the disclosedtherapeutic agents are provided in advance of any symptom, for examplein advance of infection. The prophylactic administration of thedisclosed therapeutic agents serves to prevent or ameliorate anysubsequent infection. Hence in some embodiments the methods involvesselecting a subject at risk for contracting MERS-CoV infection, andadministering a therapeutically effective amount of a disclosedimmunogen (e.g., MERS-CoV S protein or immunogenic fragment thereof,polynucleotides encoding same, protein nanoparticles, viral-likeparticles, or vectors) to the subject. The therapeutic agents can thusbe provided prior to the anticipated exposure to MERS-CoV so as toattenuate the anticipated severity, duration or extent of an infectionand/or associated disease symptoms, after exposure or suspected exposureto the virus, or after the actual initiation of an infection.

When provided therapeutically, the disclosed immunogens are provided ator after the onset of a symptom of disease or infection, for exampleafter development of a symptom of MERS-CoV infection, or after diagnosisof MERS-CoV infection. Treatment of MERS-CoV by inhibiting MERS-CoVreplication or infection can include delaying and/or reducing signs orsymptoms of MERS-CoV infection in a subject. In some examples, treatmentusing the methods disclosed herein prolongs the time of survival of thesubject.

The immunogenic composition including one or more of the disclosedagents (e.g., MERS-CoV S protein or immunogenic fragment thereof,polynucleotides encoding same, protein nanoparticles, viral-likeparticles, or vectors) can be used in coordinate vaccination protocolsor combinatorial formulations. In certain embodiments, novelcombinatorial immunogenic compositions and coordinate immunizationprotocols employ separate immunogens or formulations, each directedtoward eliciting an anti-MERS-CoV immune response, such as an immuneresponse to MERS-CoV S protein. Separate immunogenic compositions thatelicit the anti-MERS-CoV immune response can be combined in a polyvalentimmunogenic composition administered to a subject in a singleimmunization step, or they can be administered separately (in monovalentimmunogenic compositions) in a coordinate immunization protocol.

In one embodiment, a suitable immunization regimen includes at least twoseparate inoculations with one or more immunogenic compositions, with asecond inoculation being administered more than about two, about threeto eight, or about four, weeks following the first inoculation. A thirdinoculation can be administered several months (such as 2-3 months, or4, 5, or 6, months) after the second inoculation, and in specificembodiments, more than about five months after the first inoculation,more than about six months to about two years after the firstinoculation, or about eight months to about one year after the firstinoculation. Periodic inoculations beyond the third are also desirableto enhance the subject's “immune memory.” The adequacy of thevaccination parameters chosen, e.g., formulation, dose, regimen and thelike, can be determined by taking aliquots of serum from the subject andassaying antibody titers during the course of the immunization program.Alternatively, the T cell populations can be monitored by conventionalmethods. In addition, the clinical condition of the subject can bemonitored for the desired effect, e.g., prevention of MERS-CoV infectionor improvement in disease state (e.g., reduction in viral load). If suchmonitoring indicates that vaccination is sub-optimal, the subject can beboosted with an additional dose of immunogenic composition, and thevaccination parameters can be modified in a fashion expected topotentiate the immune response. Thus, for example, the dose of thedisclosed immunogen (e.g., MERS-CoV S protein or immunogenic fragmentthereof, polynucleotides encoding same, protein nanoparticles,viral-like particles, or vectors) can be increased or the route ofadministration can be changed.

It is contemplated that there can be several boosts, and that each boostcan be a different disclosed immunogen (e.g., MERS-CoV S protein orimmunogenic fragment thereof, polynucleotides encoding same, proteinnanoparticles, viral-like particles, or vectors). It is alsocontemplated in some examples that the boost may be the same immunogen(e.g., MERS-CoV S protein or immunogenic fragment thereof,polynucleotides encoding same, protein nanoparticles, viral-likeparticles, or vectors) as another boost, or the prime.

The prime and boost can be administered as a single dose or multipledoses, for example two doses, three doses, four doses, five doses, sixdoses or more can be administered to a subject over days, weeks ormonths. Multiple boosts can also be given, such one to five (e.g., 1, 2,3, 4 or 5 boosts), or more. Different dosages can be used in a series ofsequential inoculations. For example a relatively large dose in aprimary inoculation and then a boost with relatively smaller doses. Theimmune response against the selected antigenic surface can be generatedby one or more inoculations of a subject.

In one example, the method can include administering a prime-boostvaccination to a subject, including administering a therapeuticallyeffective amount of a nucleic acid molecule encoding a MERS-CoV Sprotein to the subject; and administering a therapeutically effectiveamount of a MERS-CoV S1 protein to the subject. For example the methodcan include administration of a prime including the nucleic acidmolecule encoding the MERS-CoV S protein and a boost including theMERS-CoV S1 protein. The method can include two or more administrationsof the nucleic acid molecule encoding the MERS-CoV S protein or theMERS-CoV S1 protein.

In a non-limiting example, the method includes a prime administrationcomprising administration of a nucleic acid molecule encoding theMERS-CoV S protein, a first boost administration comprisingadministration of a nucleic acid molecule encoding the MERS-CoV Sprotein, and a second boost administration including administration of aMERS-CoV S1 protein, to the subject.

In some embodiments, the prime and/or the first boost can includeadministration of a nucleic acid molecule encoding a MERS-CoV S proteincomprising or consisting of the amino acid sequence set forth as SEQ IDNO: 14, or an amino acid sequence at least 80% (such as at least 85%, atleast 90%, at least 95%, at least 98%) identical to SEQ ID NO: 14. Inmore embodiments, the second boost can include administration of aMERS-CoV S1 protein comprising or consisting of the amino acid sequenceset forth as SEQ ID NO: 16, or an amino acid sequence at least 80% (suchas at least 85%, at least 90%, at least 95%, at least 98%) identical toSEQ ID NO: 16. In more embodiments, the prime and/or the first boost caninclude administration of a nucleic acid molecule encoding a MERS-CoV Sprotein comprising or consisting of the amino acid sequence set forth asSEQ ID NO: 14, or an amino acid sequence at least 80% (such as at least85%, at least 90%, at least 95%, at least 98%) identical to SEQ ID NO:14, and the second boost can include administration of a MERS-CoV S1protein comprising or consisting of the amino acid sequence set forth asSEQ ID NO: 16, or an amino acid sequence at least 80% (such as at least85%, at least 90%, at least 95%, at least 98%) identical to SEQ ID NO:16.

Upon administration of a disclosed immunogen (e.g., MERS-CoV S proteinor immunogenic fragment thereof, polynucleotides encoding same, proteinnanoparticles, viral-like particles, or vectors) of this disclosure, theimmune system of the subject typically responds to the immunogeniccomposition by producing antibodies specific for MERS-CoV S protein.Such a response signifies that an immunologically effective dose wasdelivered to the subject.

An immunologically effective dosage can be achieved by single ormultiple administrations (including, for example, multipleadministrations per day), daily, or weekly administrations of theimmunogen. For each particular subject, specific dosage regimens can beevaluated and adjusted over time according to the individual need andprofessional judgment of the person administering or supervising theadministration of the immunogenic composition. In some embodiments, theantibody response of a subject will be determined in the context ofevaluating effective dosages/immunization protocols. In most instancesit will be sufficient to assess the antibody titer in serum or plasmaobtained from the subject. Decisions as to whether to administer boosterinoculations and/or to change the amount of the therapeutic agentadministered to the individual can be at least partially based on theantibody titer level. The antibody titer level can be based on, forexample, an immunobinding assay which measures the concentration ofantibodies in the serum which bind to an antigen including, for example,a MERS-CoV S protein. The methods of using immunogenic compositions, andthe related compositions and methods of the disclosure are also usefulin increasing resistance to, preventing, ameliorating, and/or treatinginfection and disease caused by MERS-CoV in animal hosts, and other, invitro applications.

In several embodiments, a disclosed immunogen can be administered to thesubject simultaneously with the administration of an adjuvant. In otherembodiments, the immunogen (e.g., MERS-CoV S protein or immunogenicfragment thereof, polynucleotides encoding same, protein nanoparticles,viral-like particles, or vectors) is administered to the subject afterthe administration of an adjuvant and within a sufficient amount of timeto induce the immune response. Non-limiting examples of adjuvantsinclude aluminum hydroxide or aluminum phosphate, TLR9 agonist (likeCpG), TLR7 and/or TLR8 agonists, TLR5 agonist (like flagellin), any TLR4agonist (any variant of lipid A), TLR3 agonist (variants ofdouble-stranded RNA like polyl:C), any oil-in-water immulsion (many ofthese use squalene), ISCOMS, or anything containing QS21, anythingcombined with lipid membranes like virosomes.

For prophylactic and therapeutic purposes, a therapeutically effectiveamount of a disclosed immunogen (e.g., MERS-CoV S protein or immunogenicfragment thereof, polynucleotides encoding same, protein nanoparticles,viral-like particles, or vectors) can be administered to the subject ina single bolus delivery, via continuous delivery (for example,continuous transdermal, mucosal or intravenous delivery) over anextended time period, or in a repeated administration protocol (forexample, by an hourly, daily or weekly, repeated administrationprotocol). The therapeutically effective dosage of the therapeuticagents can be provided as repeated doses within a prolonged prophylaxisor treatment regimen that will yield clinically significant results toalleviate one or more symptoms or detectable conditions associated witha targeted disease or condition as set forth herein.

Determination of effective dosages in this context is typically based onanimal model studies followed up by human clinical trials and is guidedby administration protocols that significantly reduce the occurrence orseverity of targeted disease symptoms or conditions in the subject, orthat induce a desired response in the subject (such as a neutralizingimmune response). Suitable models in this regard include, for example,murine, rat, porcine, feline, ferret, non-human primate, and otheraccepted animal model subjects known in the art. Alternatively,effective dosages can be determined using in vitro models (for example,immunologic and histopathologic assays). Using such models, onlyordinary calculations and adjustments are required to determine anappropriate concentration and dose to administer a therapeuticallyeffective amount of the composition (for example, amounts that areeffective to elicit a desired immune response or alleviate one or moresymptoms of a targeted disease). In alternative embodiments, aneffective amount or effective dose of the composition may simply inhibitor enhance one or more selected biological activities correlated with adisease or condition, as set forth herein, for either therapeutic ordiagnostic purposes.

Dosage can be varied by the attending clinician to maintain a desiredconcentration at a target site (for example, systemic circulation).Higher or lower concentrations can be selected based on the mode ofdelivery, for example, trans-epidermal, rectal, oral, pulmonary, orintranasal delivery versus intravenous or subcutaneous delivery. Theactual dosage of disclosed immunogen (e.g., MERS-CoV S protein orimmunogenic fragment thereof, polynucleotides encoding same, proteinnanoparticles, viral-like particles, or vectors) will vary according tofactors such as the disease indication and particular status of thesubject (for example, the subject's age, size, fitness, extent ofsymptoms, susceptibility factors, and the like), time and route ofadministration, other drugs or treatments being administeredconcurrently, as well as the specific pharmacology of the compositionfor eliciting the desired activity or biological response in thesubject. Dosage regimens can be adjusted to provide an optimumprophylactic or therapeutic response. As described above in the forgoinglisting of terms, a therapeutically effective amount is also one inwhich any toxic or detrimental side effects of the disclosed immunogenand/or other biologically active agent is outweighed in clinical termsby therapeutically beneficial effects.

A non-limiting range for a therapeutically effective amount of thedisclosed polypeptide immunogen (e.g., MERS-CoV S protein or immunogenicfragment thereof, polynucleotides encoding same, protein nanoparticles,viral-like particles, or vectors) within the methods and immunogeniccompositions of the disclosure is about 0.0001 mg/kg body weight toabout 10 mg/kg body weight, such as about 0.01 mg/kg, about 0.02 mg/kg,about 0.03 mg/kg, about 0.04 mg/kg, about 0.05 mg/kg, about 0.06 mg/kg,about 0.07 mg/kg, about 0.08 mg/kg, about 0.09 mg/kg, about 0.1 mg/kg,about 0.2 mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg,about 0.6 mg/kg, about 0.7 mg/kg, about 0.8 mg/kg, about 0.9 mg/kg,about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3mg/kg, about 4 mg/kg, about 5 mg/kg, or about 10 mg/kg, for example 0.01mg/kg to about 1 mg/kg body weight, about 0.05 mg/kg to about 5 mg/kgbody weight, about 0.2 mg/kg to about 2 mg/kg body weight, or about 1.0mg/kg to about 10 mg/kg body weight.

In some embodiments, the dosage includes a set amount of a disclosedimmunogen (e.g., MERS-CoV S protein or immunogenic fragment thereof,polynucleotides encoding same, protein nanoparticles, viral-likeparticles, or vectors) such as from about 1-300 μg, for example, adosage of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60,70, 80, 90, 100, 150, 200, 250, or about 300 μg.

The dosage and number of doses will depend on the setting, for example,in an adult or anyone primed by prior MERS-CoV infection orimmunization, a single dose may be a sufficient booster. In naïvesubjects, in some examples, at least two doses would be given, forexample, at least three doses. In some embodiments, an annual boost isgiven, for example, along with an annual influenza vaccination.

Actual methods for preparing administrable compositions will be known orapparent to those skilled in the art and are described in more detail insuch publications as Remingtons Pharmaceutical Sciences, 19^(th) Ed.,Mack Publishing Company, Easton, Pa., 1995.

Administration of a therapeutically effective amount of a disclosedimmunogen (e.g., MERS-CoV S protein or immunogenic fragment thereof,polynucleotides encoding same, protein nanoparticles, viral-likeparticles, or vectors) induces a sufficient immune response to treat orinhibit or prevent the pathogenic infection, for example, to inhibit theinfection and/or reduce the signs and/or symptoms of the infection.Amounts effective for this use will depend upon the severity of thedisease, the general state of the subject's health, and the robustnessof the subject's immune system.

MERS-CoV infection does not need to be completely eliminated or reducedor prevented for the methods to be effective. For example, treatmentwith one or more of the disclosed therapeutic agents can reduce orinhibit MERS-CoV infection by a desired amount, for example by at least10%, at least 20%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 95%, at least 98%, or even at least 100%(elimination or prevention of detectable MERS-CoV infected cells), ascompared to MERS-CoV infection in the absence of the therapeutic agent.In additional examples, MERS-CoV replication can be reduced or inhibitedby the disclosed methods. MERS-CoV replication does not need to becompletely eliminated for the method to be effective. For example,treatment with one or more of the disclosed immunogens (e.g., MERS-CoV Sprotein or immunogenic fragment thereof, polynucleotides encoding same,protein nanoparticles, viral-like particles, or vectors) can reduceMERS-CoV replication by a desired amount, for example by at least 10%,at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, or even at least 100%(elimination or prevention of detectable MERS-CoV replication), ascompared to MERS-CoV replication in the absence of the therapeuticagent.

In several embodiments, following immunization of a subject with adisclosed immunogen, serum can be collected from the subject atappropriate time points, frozen, and stored for neutralization testing.Methods to assay for neutralization activity are known to the person ofordinary skill in the art and are further described herein, and include,but are not limited to, plaque reduction neutralization (PRNT) assays,microneutralization assays, flow cytometry based assays, single-cycleinfection assays, and pseudovirus neutralization assays (e.g., asdescribed in Example 1).

In some embodiments, administration of a therapeutically effectiveamount of one or more of the disclosed immunogens to a subject (e.g., bya prime-boost administration of a DNA vector encoding a disclosedimmunogen (prime) followed by a MERS-CoV S protein or fragment thereof(such as an S1 protein) or a protein nanoparticle including the MERS-CoVS protein or fragment thereof (boost)) induces a neutralizing immuneresponse in the subject. In several embodiments, the neutralizing immuneresponse can be detected using a pseudovirus neutralization assayagainst a panel of MERS-CoV pseudoviruses including MERS-CoV S proteinsfrom different MERS-CoV strains, for example, as described in Example 1.In some embodiments, administration of the therapeutically effectiveamount of disclosed immunogens to a subject by the prime-boostadministration of a DNA vector encoding a disclosed immunogen (prime)followed by a MERS-CoV S protein or fragment thereof (such as an S1protein) or a protein nanoparticle including the MERS-CoV S protein orfragment thereof (boost) induces a neutralizing immune response in thesubject, wherein serum from the subject neutralizes, with an ID₅₀≥40, atleast 30% (such as at least 40%, at least 50%, at least 60%, at least70%, at least 80%, or at least 90%) of pseudoviruses in a panel ofpseudoviruses including the MERS-CoV S proteins listed in FIG. 1C.

One approach to administration of nucleic acids is direct immunizationwith plasmid DNA, such as with a mammalian expression plasmid. Asdescribed above, the nucleotide sequence encoding a disclosedrecombinant MERS-CoV S protein or fragment thereof can be placed underthe control of a promoter to increase expression of the molecule.

Immunization by nucleic acid constructs is well known in the art andtaught, for example, in U.S. Pat. No. 5,643,578 (which describes methodsof immunizing vertebrates by introducing DNA encoding a desired antigento elicit a cell-mediated or a humoral response), and U.S. Pat. Nos.5,593,972 and 5,817,637 (which describe operably linking a nucleic acidsequence encoding an antigen to regulatory sequences enablingexpression). U.S. Pat. No. 5,880,103 describes several methods ofdelivery of nucleic acids encoding immunogenic peptides or otherantigens to an organism. The methods include liposomal delivery of thenucleic acids (or of the synthetic peptides themselves), andimmune-stimulating constructs, or ISCOMS™, negatively charged cage-likestructures of 30-40 nm in size formed spontaneously on mixingcholesterol and Quil A™ (saponin). Immunization with RNA-basedtechnology can also be used with the disclosed embodiments. Protectiveimmunity has been generated in a variety of experimental models ofinfection, including toxoplasmosis and Epstein-Barr virus-inducedtumors, using ISCOMS™ as the delivery vehicle for antigens (Mowat andDonachie, Immunol. Today 12:383, 1991). Doses of antigen as low as 1 μgencapsulated in ISCOMS™ have been found to produce Class I mediated CTLresponses (Takahashi et al., Nature 344:873, 1990).

In another approach to using nucleic acids for immunization, a disclosedMERS-CoV S protein or fragment thereof, can also be expressed byattenuated viral hosts or vectors or bacterial vectors. Recombinantvaccinia virus, adeno-associated virus (AAV), herpes virus, retrovirus,cytomegalovirus, alphavirus (e.g., as described in Lundstrom, Viruses,7(5):2321-2333, 2015), or other viral vectors can be used to express thepeptide or protein, thereby eliciting a CTL response. For example,vaccinia vectors and methods useful in immunization protocols aredescribed in U.S. Pat. No. 4,722,848. BCG (Bacillus Calmette Guerin)provides another vector for expression of the peptides (see Stover,Nature 351:456-460, 1991).

In one embodiment, a nucleic acid encoding a disclosed MERS-CoV Sprotein or fragment thereof, is introduced directly into cells. Forexample, the nucleic acid can be loaded onto gold microspheres bystandard methods and introduced into the skin by a device such asBio-Rad's HELIOS™ Gene Gun. The nucleic acids can be “naked,” consistingof plasmids under control of a strong promoter. Typically, the DNA isinjected into muscle, although it can also be injected directly intoother sites. Dosages for injection are usually around 0.5 Kg/kg to about50 mg/kg, and typically are about 0.005 mg/kg to about 5 mg/kg (see,e.g., U.S. Pat. No. 5,589,466).

In certain embodiments, the immunogen can be administered sequentiallywith other anti-MERS-CoV therapeutic agents, such as before or after theother agent. One of ordinary skill in the art would know that sequentialadministration can mean immediately following or after an appropriateperiod of time, such as hours, days, weeks, months, or even years later.

IV. Polynucleotides and Expression

Polynucleotides encoding a disclosed MERS-CoV S protein or immunogenicfragment thereof, or protein nanoparticles (or a subunit thereof), or anantibody, antibody binding fragment, or conjugate that specificallybinds MERS-CoV S protein are also provided. These polynucleotidesinclude DNA, cDNA and RNA sequences which encode the disclosed MERS-CoVS protein or immunogenic fragment thereof, or protein nanoparticle (or asubunit thereof), or an antibody, antibody binding fragment, orconjugate that specifically binds MERS-CoV S protein. Nucleic acidsencoding these molecules can readily be produced by one of skill in theart, using the amino acid sequences provided herein (such as the CDR andheavy chain and light chain sequences for production of antibodies),sequences available in the art (such as framework sequences), and thegenetic code. One of skill in the art can readily use the genetic codeto construct a variety of functionally equivalent nucleic acids, such asnucleic acids which differ in sequence but which encode the sameantibody sequence, or encode a conjugate or fusion protein including thenucleic acid sequence.

Polynucleotides encoding a disclosed MERS-CoV S protein or immunogenicfragment thereof, or protein nanoparticles (or a subunit thereof), or anantibody, antibody binding fragment, or conjugate that specificallybinds MERS-CoV S protein can be prepared by any suitable methodincluding, for example, cloning of appropriate sequences or by directchemical synthesis by methods such as the phosphotriester method ofNarang et al., Meth. Enzymol. 68:90-99, 1979; the phosphodiester methodof Brown et al., Meth. Enzymol. 68:109-151, 1979; thediethylphosphoramidite method of Beaucage et al., Tetra. Lett.22:1859-1862, 1981; the solid phase phosphoramidite triester methoddescribed by Beaucage & Caruthers, Tetra. Letts. 22(20):1859-1862, 1981,for example, using an automated synthesizer as described in, forexample, Needham-VanDevanter et al., NucL Acids Res. 12:6159-6168, 1984;and, the solid support method of U.S. Pat. No. 4,458,066. Chemicalsynthesis produces a single stranded oligonucleotide. This can beconverted into double stranded DNA by hybridization with a complementarysequence or by polymerization with a DNA polymerase using the singlestrand as a template. One of skill would recognize that while chemicalsynthesis of DNA is generally limited to sequences of about 100 bases,longer sequences may be obtained by the ligation of shorter sequences.

Examples of appropriate cloning and sequencing techniques, andinstructions sufficient to direct persons of skill through many cloningexercises are known (see, e.g, Sambrook et al. (Molecular Cloning: ALaboratory Manual, 4^(th) ed, Cold Spring Harbor, N.Y., 2012) andAusubel et al. (In Current Protocols in Molecular Biology, John Wiley &Sons, New York, through supplement 104, 2013). Product information frommanufacturers of biological reagents and experimental equipment alsoprovide useful information. Such manufacturers include the SIGMAChemical Company (Saint Louis, Mo.), R&D Systems (Minneapolis, Minn.),Pharmacia Amersham (Piscataway, N.J.), CLONTECH Laboratories, Inc. (PaloAlto, Calif.), Chem Genes Corp., Aldrich Chemical Company (Milwaukee,Wis.), Glen Research, Inc., GIBCO BRL Life Technologies, Inc.(Gaithersburg, Md.), Fluka Chemica-Biochemika Analytika (Fluka ChemieAG, Buchs, Switzerland), Invitrogen (Carlsbad, Calif.), and AppliedBiosystems (Foster City, Calif.), as well as many other commercialsources known to one of skill.

Nucleic acids can also be prepared by amplification methods.Amplification methods include polymerase chain reaction (PCR), theligase chain reaction (LCR), the transcription-based amplificationsystem (TAS), the self-sustained sequence replication system (3SR). Awide variety of cloning methods, host cells, and in vitro amplificationmethodologies are well known to persons of skill.

The nucleic acid molecules can be expressed in a recombinantlyengineered cell such as bacteria, plant, yeast, insect and mammaliancells. Methods of expressing DNA sequences having eukaryotic or viralsequences in prokaryotes are well known in the art. Non-limitingexamples of suitable host cells include bacteria, archea, insect, fungi(for example, yeast), plant, and animal cells (for example, mammaliancells, such as human). Exemplary cells of use include Escherichia coli,Bacillus subtilis, Saccharomyces cerevisiae, Salmonella typhimurium, SF9cells, C129 cells, 293 cells, Neurospora, and immortalized mammalianmyeloid and lymphoid cell lines. Techniques for the propagation ofmammalian cells in culture are well-known (see, e.g., Helgason andMiller (Eds.), 2012, Basic Cell Culture Protocols (Methods in MolecularBiology), 4^(th) Ed., Humana Press). Examples of commonly used mammalianhost cell lines are VERO and HeLa cells, CHO cells, and WI38, BHK, andCOS cell lines, although cell lines may be used, such as cells designedto provide higher expression, desirable glycosylation patterns, or otherfeatures. In some embodiments, the host cells include HEK293 cells orderivatives thereof, such as GnTI^(−/−) cells (ATCC® No. CRL-3022), orHEK-293F cells.

The expression of nucleic acids encoding the proteins described hereincan be achieved by operably linking the DNA or cDNA to a promoter (whichis either constitutive or inducible), followed by incorporation into anexpression cassette. The promoter can be any promoter of interest,including a cytomegalovirus promoter and a human T cell lymphotrophicvirus promoter (HTLV)-1. Optionally, an enhancer, such as acytomegalovirus enhancer, is included in the construct. The cassettescan be suitable for replication and integration in either prokaryotes oreukaryotes. Typical expression cassettes contain specific sequencesuseful for regulation of the expression of the DNA encoding the protein.For example, the expression cassettes can include appropriate promoters,enhancers, transcription and translation terminators, initiationsequences, a start codon (i.e., ATG) in front of a protein-encodinggene, splicing signal for introns, sequences for the maintenance of thecorrect reading frame of that gene to permit proper translation of mRNA,and stop codons. The vector can encode a selectable marker, such as amarker encoding drug resistance (for example, ampicillin or tetracyclineresistance).

To obtain high level expression of a cloned gene, it is desirable toconstruct expression cassettes which contain, at the minimum, a strongpromoter to direct transcription, a ribosome binding site fortranslational initiation (internal ribosomal binding sequences), and atranscription/translation terminator. For E. coli, this includes apromoter such as the T7, trp, lac, or lambda promoters, a ribosomebinding site, and preferably a transcription termination signal. Foreukaryotic cells, the control sequences can include a promoter and/or anenhancer derived from, for example, an immunoglobulin gene, HTLV, SV40or cytomegalovirus, and a polyadenylation sequence, and can furtherinclude splice donor and/or acceptor sequences (for example, CMV and/orHTLV splice acceptor and donor sequences). The cassettes can betransferred into the chosen host cell by well-known methods such astransformation or electroporation for E. coli and calcium phosphatetreatment, electroporation or lipofection for mammalian cells. Cellstransformed by the cassettes can be selected by resistance toantibiotics conferred by genes contained in the cassettes, such as theamp, gpt, neo and hyg genes.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate coprecipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransformed with polynucleotide sequences encoding a disclosedMERS-CoV S protein or immunogenic fragment thereof, or proteinnanoparticles (or a subunit thereof), or an antibody, antibody bindingfragment, or conjugate that specifically binds MERS-CoV S protein, and asecond foreign DNA molecule encoding a selectable phenotype, such as theherpes simplex thymidine kinase gene. Another method is to use aeukaryotic viral vector, such as simian virus 40 (SV40) or bovinepapilloma virus, to transiently infect or transform eukaryotic cells andexpress the protein (see for example, Viral Expression Vectors, Springerpress, Muzyczka ed., 2011). One of skill in the art can readily use anexpression systems such as plasmids and vectors of use in producingproteins in cells including higher eukaryotic cells such as the COS,CHO, HeLa and myeloma cell lines.

The antibodies, antigen binding fragments, and conjugates can beexpressed as individual V_(H) and/or V_(L) chain (linked to an effectormolecule or detectable marker as needed), or can be expressed as afusion protein. Methods of expressing and purifying antibodies andantigen binding fragments are known and further described herein (see,e.g., Al-Rubeai (ed), Antibody Expression and Production, SpringerPress, 2011). An immunoadhesin can also be expressed. Thus, in someexamples, nucleic acids encoding a V_(H) and V_(L), and immunoadhesinare provided. The nucleic acid sequences can optionally encode a leadersequence.

To create a scFv the V_(H)- and V_(L)-encoding DNA fragments can beoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly4-Ser)₃, such that the V_(H) andV_(L) sequences can be expressed as a contiguous single-chain protein,with the V_(L) and V_(H) domains joined by the flexible linker (see,e.g., Bird et al., Science 242:423-426, 1988; Huston et al., Proc. Natl.Acad. Sci. USA 85:5879-5883, 1988; McCafferty et al., Nature348:552-554, 1990; Kontermann and Dubel (Ed), Antibody Engineering,Vols. 1-2, 2^(nd) Ed., Springer Press, 2010; Harlow and Lane,Antibodies: A Laboratory Manual, 2^(nd), Cold Spring Harbor Laboratory,New York, 2013,). Optionally, a cleavage site can be included in alinker, such as a furin cleavage site.

The nucleic acid encoding a V_(H) and/or the V_(L) optionally can encodean Fc domain (immunoadhesin). The Fc domain can be an IgA, IgM or IgG Fcdomain. The Fc domain can be an optimized Fc domain, as described inU.S. Published Patent Application No. 20100/093979, incorporated hereinby reference. In one example, the immunoadhesin is an IgG₁ Fc.

The single chain antibody may be monovalent, if only a single V_(H) andV_(L) are used, bivalent, if two V_(H) and V_(L) are used, orpolyvalent, if more than two V_(H) and V_(L) are used. Bispecific orpolyvalent antibodies may be generated that bind specifically toMERS-CoV S protein and another antigen, such as, but not limited to CD3.The encoded V_(H) and V_(L) optionally can include a furin cleavage sitebetween the V_(H) and V_(L) domains.

Methods for expression of antibodies, antigen binding fragments, andconjugates, and/or refolding to an appropriate active form, frommammalian cells, and bacteria such as E. coli have been described andare well-known and are applicable to the antibodies disclosed herein.See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, 2^(nd),Cold Spring Harbor Laboratory, New York, 2013, Simpson ed., Basicmethods in Protein Purification and Analysis: A laboratory Manual, ColdHarbor Press, 2008, and Ward et al., Nature 341:544, 1989.

Also provided is a population of cells comprising at least one host celldescribed herein. The population of cells can be a heterogeneouspopulation comprising the host cell comprising any of the recombinantexpression vectors described, in addition to at least one other cell,e.g., a host cell (e.g., a T cell), which does not comprise any of therecombinant expression vectors, or a cell other than a T cell, e.g., a Bcell, a macrophage, a neutrophil, an erythrocyte, a hepatocyte, anendothelial cell, an epithelial cell, a muscle cell, a brain cell, etc.Alternatively, the population of cells can be a substantiallyhomogeneous population, in which the population comprises mainly hostcells (e.g., consisting essentially of) comprising the recombinantexpression vector. The population also can be a clonal population ofcells, in which all cells of the population are clones of a single hostcell comprising a recombinant expression vector, such that all cells ofthe population comprise the recombinant expression vector. In oneembodiment of the invention, the population of cells is a clonalpopulation comprising host cells comprising a recombinant expressionvector as described herein

Modifications can be made to a nucleic acid encoding a polypeptidedescribed herein without diminishing its biological activity. Somemodifications can be made to facilitate the cloning, expression, orincorporation of the targeting molecule into a fusion protein. Suchmodifications are well known to those of skill in the art and include,for example, termination codons, a methionine added at the aminoterminus to provide an initiation, site, additional amino acids placedon either terminus to create conveniently located restriction sites, oradditional amino acids (such as poly His) to aid in purification steps.In addition to recombinant methods, the immunoconjugates, effectormoieties, and antibodies of the present disclosure can also beconstructed in whole or in part using standard peptide synthesis wellknown in the art.

In several embodiments, the nucleic acid molecule encodes a precursor ofa disclosed MERS-CoV S protein or fragment thereof that can be processedinto the MERS-CoV S protein or fragment thereof when expressed in anappropriate cell. For example, the nucleic acid molecule can encode aMERS-CoV S protein or fragment thereof including a N-terminal signalsequence for entry into the cellular secretory system that isproteolytically cleaved in the during processing of the MERS-CoV Sprotein or fragment in the cell. In some embodiments, the signal peptideincludes the amino acid sequence set forth as SEQ ID NOs: 24 or 25.

The polynucleotides encoding a MERS-CoV S protein or fragment thereof,or protein nanoparticle subunit linked to such a S protein or fragmentcan include a recombinant DNA which is incorporated into a vector intoan autonomously replicating plasmid or virus or into the genomic DNA ofa prokaryote or eukaryote, or which exists as a separate molecule (suchas an mRNA or a cDNA) independent of other sequences. The nucleotidescan be ribonucleotides, deoxyribonucleotides, or modified forms ofeither nucleotide. The term includes single and double forms of DNA. Inone non-limiting example, a disclosed immunogen is expressed using thepVRC8400 vector (described in Barouch et al., J. Virol, 79, 8828-8834,2005, which is incorporated by reference herein).

Once expressed, a disclosed MERS-CoV S protein or immunogenic fragmentthereof, or protein nanoparticles (or a subunit thereof), or anantibody, antibody binding fragment, or conjugate that specificallybinds MERS-CoV S protein can be purified according to standardprocedures in the art, including ammonium sulfate precipitation,affinity columns, column chromatography, and the like (see, generally,Simpson ed., Basic methods in Protein Purification and Analysis: Alaboratory Manual, Cold Harbor Press, 2008). The MERS-CoV S protein orimmunogenic fragment thereof, or protein nanoparticles (or a subunitthereof), or an antibody, antibody binding fragment, or conjugate thatspecifically binds MERS-CoV S protein need not be 100% pure. Oncepurified, partially or to homogeneity as desired, if to be usedtherapeutically, the polypeptides should be substantially free ofendotoxin.

Often, functional heterologous proteins from E. coli or other bacteriaare isolated from inclusion bodies and require solubilization usingstrong denaturants, and subsequent refolding. During the solubilizationstep, as is well known in the art, a reducing agent must be present toseparate disulfide bonds. An exemplary buffer with a reducing agent is:0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol).Reoxidation of the disulfide bonds can occur in the presence of lowmolecular weight thiol reagents in reduced and oxidized form, asdescribed in Saxena et al., Biochemistry 9: 5015-5021, 1970, andespecially as described by Buchner et al., supra.

In addition to recombinant methods, the antibodies, antigen bindingfragments, and/or conjugates can also be constructed in whole or in partusing standard peptide synthesis. Solid phase synthesis of thepolypeptides can be accomplished by attaching the C-terminal amino acidof the sequence to an insoluble support followed by sequential additionof the remaining amino acids in the sequence. Techniques for solid phasesynthesis are described by Barany & Merrifield, The Peptides: Analysis,Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, PartA. pp. 3-284; Merrifield et al., J. Am. Chem. Soc. 85:2149-2156, 1963,and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem.Co., Rockford, Ill., 1984. Proteins of greater length may be synthesizedby condensation of the amino and carboxyl termini of shorter fragments.Methods of forming peptide bonds by activation of a carboxyl terminalend (such as by the use of the coupling reagentN,N′-dicylohexylcarbodimide) are well known in the art.

V. Compositions and Administration

The disclosed agents can be included in a pharmaceutical composition(including therapeutic and prophylactic formulations), often combinedtogether with one or more pharmaceutically acceptable vehicles and,optionally, other therapeutic ingredients (for example, antibiotics orantiviral drugs). In several embodiments, pharmaceutical compositionsincluding one or more of the disclosed immunogens are immunogeniccompositions. The compositions are useful, for example, for example, forthe treatment or detection of a MERS-CoV infection or induction of animmune response to MERS-CoV in a subject.

The compositions can be prepared in unit dosage forms for administrationto a subject. The amount and timing of administration are at thediscretion of the treating physician to achieve the desired purposes. Adisclosed MERS-CoV S protein or immunogenic fragment thereof, or proteinnanoparticle (or a subunit thereof), or an antibody, antibody bindingfragment, or conjugate that specifically binds MERS-CoV S protein, orpolynucleotide encoding such molecules can be formulated for systemic orlocal administration. In one example, the disclosed MERS-CoV S proteinor immunogenic fragment thereof, or protein nanoparticle (or a subunitthereof), or an antibody, antibody binding fragment, or conjugate thatspecifically binds MERS-CoV S protein, or polynucleotide encoding suchmolecules is formulated for parenteral administration, such asintravenous administration.

A disclosed MERS-CoV S protein or immunogenic fragment thereof, orprotein nanoparticle (or a subunit thereof), or an antibody, antibodybinding fragment, or conjugate that specifically binds MERS-CoV Sprotein, or polynucleotide encoding such molecules, or a compositionincluding such molecules, as well as additional agents, can beadministered to subjects in various ways, including local and systemicadministration, such as, e.g., by injection subcutaneously,intravenously, intra-arterially, intranasally, intraperitoneally,intramuscularly, intradermally, or intrathecally. In an embodiment, atherapeutic agent is administered by a single subcutaneous, intravenous,intra-arterial, intraperitoneal, intramuscular, intradermal orintrathecal injection once a day. The therapeutic agent can also beadministered by direct injection at or near the site of disease.

A further method of administration is by osmotic pump (e.g., an Alzetpump) or mini-pump (e.g., an Alzet mini-osmotic pump), which allows forcontrolled, continuous and/or slow-release delivery of the therapeuticagent or pharmaceutical composition over a pre-determined period. Theosmotic pump or mini-pump can be implanted subcutaneously, or near atarget site.

The therapeutic agent or compositions thereof can also be administeredby other modes. Determination of the most effective mode ofadministration of the therapeutic agent or compositions thereof iswithin the skill of the skilled artisan. The therapeutic agent can beadministered as pharmaceutical formulations suitable for, e.g., oral(including buccal and sub-lingual), rectal, nasal, topical, pulmonary,vaginal or parenteral administration, or in a form suitable foradministration by inhalation or insufflation. Depending on the intendedmode of administration, the pharmaceutical formulations can be in theform of solid, semi-solid or liquid dosage forms, such as tablets,suppositories, pills, capsules, powders, liquids, suspensions,emulsions, creams, ointments, lotions, and the like.

In some embodiments, the compositions comprise a disclosed MERS-CoV Sprotein or immunogenic fragment thereof, or protein nanoparticle (or asubunit thereof), or an antibody, antibody binding fragment, orconjugate that specifically binds MERS-CoV S protein, or polynucleotideencoding such molecules in at least about 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98% or 99% purity. In certain embodiments, the compositionscontain less than about 10%, 5%, 4%, 3%, 2%, 1% or 0.5% ofmacromolecular contaminants, such as other mammalian (e.g., human)proteins.

In some embodiments, the composition can be provided in unit dosage formfor use to induce an immune response in a subject, for example, toprevent, inhibit, or treat MERS-CoV infection in the subject. A unitdosage form contains a suitable single preselected dosage foradministration to a subject, or suitable marked or measured multiples oftwo or more preselected unit dosages, and/or a metering mechanism foradministering the unit dose or multiples thereof. In other embodiments,the composition further includes an adjuvant.

A typical composition for intravenous administration of an antibody orantigen binding fragment thereof includes about 0.01 to about 30 mg/kgof antibody or antigen binding fragment or conjugate per subject per day(or the corresponding dose of a conjugate including the antibody orantigen binding fragment). Actual methods for preparing administrablecompositions will be known or apparent to those skilled in the art andare described in more detail in such publications as Remington'sPharmaceutical Science, 19th ed., Mack Publishing Company, Easton, Pa.(1995). In some embodiments, the composition can be a liquid formulationincluding one or more antibodies, antigen binding fragments (such as anantibody or antigen binding fragment that specifically binds to MERS-CoVS protein), in a concentration range from about 0.1 mg/ml to about 20mg/ml, or from about 0.5 mg/ml to about 20 mg/ml, or from about 1 mg/mlto about 20 mg/ml, or from about 0.1 mg/ml to about 10 mg/ml, or fromabout 0.5 mg/ml to about 10 mg/ml, or from about 1 mg/ml to about 10mg/ml.

An antibody solution, or an antigen binding fragment can be added to aninfusion bag containing 0.9% sodium chloride, USP, and typicallyadministered at a dosage of from 0.5 to 15 mg/kg of body weight.Considerable experience is available in the art in the administration ofantibody drugs, which have been marketed in the U.S. since the approvalof RITUXAN® in 1997. Antibodies and antigen binding fragments can beadministered by slow infusion, rather than in an intravenous push orbolus. In one example, a higher loading dose is administered, withsubsequent, maintenance doses being administered at a lower level. Forexample, an initial loading dose of 4 mg/kg may be infused over a periodof some 90 minutes, followed by weekly maintenance doses for 4-8 weeksof 2 mg/kg infused over a 30 minute period if the previous dose was welltolerated.

In several embodiments, an immunogenic composition (e.g., including adisclosed MERS-CoV S protein or immunogenic fragment thereof, or proteinnanoparticle (or a subunit thereof) or polynucleotide encoding suchmolecules) include an adjuvant. The person of ordinary skill in the artis familiar with adjuvants, for example, those that can be included inan immunogenic composition. It will be appreciated that the choice ofadjuvant can be different in these different applications, and theoptimal adjuvant and concentration for each situation can be determinedempirically by those of skill in the art. Adjuvants, such as aluminumhydroxide (ALHYDROGEL®, available from Brenntag Biosector, Copenhagen,Denmark and AMPHOGEL®, Wyeth Laboratories, Madison, N.J.), Freund'sadjuvant, MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton,Ind.) and IL-12 (Genetics Institute, Cambridge, Mass.), among many othersuitable adjuvants well known in the art, can be included in thecompositions.

Preparation of immunogenic compositions, including those foradministration to human subjects, is known in the art, and generallydescribed, for example, in Pharmaceutical Biotechnology, Vol. 61 VaccineDesign-the subunit and adjuvant approach, edited by Powell and Newman,Plenum Press, 1995. New Trends and Developments in Vaccines, edited byVoller et al., University Park Press, Baltimore, Md., U.S.A. 1978.Encapsulation within liposomes is described, for example, by Fullerton,U.S. Pat. No. 4,235,877. Conjugation of proteins to macromolecules isdisclosed, for example, by Likhite, U.S. Pat. No. 4,372,945 and by Armoret al., U.S. Pat. No. 4,474,757. Typically, the amount of antigen ineach dose of the immunogenic composition is selected as an amount whichinduces an immune response without significant, adverse side effects.

The amount of the disclosed immunogen (for example, disclosed MERS-CoV Sprotein or immunogenic fragment thereof, or protein nanoparticle (or asubunit thereof), or viral vector or polynucleotide encoding suchmolecules, included in the immunogenic composition can vary dependingupon the specific antigen employed, the route and protocol ofadministration, and the target population, for example. For proteintherapeutics, typically, each human dose will comprise 1-1000 μg ofprotein, such as from about 1 μg to about 100 μg, for example, fromabout 1 μg to about 50 μg, such as about 1 μg, about 2 μg, about 5 μg,about 10 μg, about 15 μg, about 20 μg, about 25 μg, about 30 μg, about40 μg, or about 50 μg. The amount utilized in an immunogenic compositioncan be selected based on the subject population (e.g., infant orelderly). An optimal amount for a particular composition can beascertained by standard studies involving observation of antibody titersand other responses in subjects. It is understood that a therapeuticallyeffective amount of a disclosed immunogen, such as disclosed MERS-CoV Sprotein or immunogenic fragment thereof, or protein nanoparticle (or asubunit thereof), or viral vector or polynucleotide encoding suchmolecules can include an amount that is ineffective at eliciting animmune response by administration of a single dose, but that iseffective upon administration of multiple dosages or in combinationadministration of a second immunogen, for example in a prime-boostadministration protocol.

To formulate the pharmaceutical compositions, the disclosed MERS-CoV Sprotein or immunogenic fragment thereof, or protein nanoparticle (or asubunit thereof), viral vector or an antibody, antibody bindingfragment, or conjugate that specifically binds MERS-CoV S protein, orpolynucleotide encoding such molecules, can be combined with variouspharmaceutically acceptable additives, as well as a base or vehicle fordispersion of the conjugate. Desired additives include, but are notlimited to, pH control agents, such as arginine, sodium hydroxide,glycine, hydrochloric acid, citric acid, and the like. In addition,local anesthetics (for example, benzyl alcohol), isotonizing agents (forexample, sodium chloride, mannitol, sorbitol), adsorption inhibitors(for example, TWEEN® 80), solubility enhancing agents (for example,cyclodextrins and derivatives thereof), stabilizers (for example, serumalbumin), and reducing agents (for example, glutathione) can beincluded.

The compositions for administration can include a solution of thedisclosed MERS-CoV S protein or immunogenic fragment thereof, or proteinnanoparticle (or a subunit thereof), viral vector or an antibody,antibody binding fragment, or conjugate that specifically binds MERS-CoVS protein, or polynucleotide encoding such molecules dissolved in apharmaceutically acceptable carrier, such as an aqueous carrier. Avariety of aqueous carriers can be used, for example, buffered salineand the like. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiological conditionssuch as pH adjusting and buffering agents, toxicity adjusting agents andthe like, for example, sodium acetate, sodium chloride, potassiumchloride, calcium chloride, sodium lactate and the like. Theconcentration of the disclosed MERS-CoV S protein or immunogenicfragment thereof, or protein nanoparticle (or a subunit thereof), viralvector or an antibody, antibody binding fragment, or conjugate thatspecifically binds MERS-CoV S protein, or polynucleotide encoding suchmolecules in these formulations can vary widely, and will be selectedprimarily based on fluid volumes, viscosities, body weight and the likein accordance with the particular mode of administration selected andthe subject's needs.

The disclosed MERS-CoV S protein or immunogenic fragment thereof, orprotein nanoparticle (or a subunit thereof), viral vector or anantibody, antibody binding fragment, or conjugate that specificallybinds MERS-CoV S protein, or polynucleotide encoding such molecules canbe provided in lyophilized form and rehydrated with sterile water beforeadministration, although they are also provided in sterile solutions ofknown concentration.

Controlled-release parenteral formulations can be made as implants, oilyinjections, or as particulate systems. For a broad overview of proteindelivery systems see, Banga, A. J., Therapeutic Peptides and Proteins:Formulation, Processing, and Delivery Systems, Technomic PublishingCompany, Inc., Lancaster, Pa., (1995). Particulate systems includemicrospheres, microparticles, microcapsules, nanocapsules, nanospheres,and nanoparticles. Microcapsules contain the therapeutic protein, suchas a cytotoxin or a drug, as a central core. In microspheres thetherapeutic is dispersed throughout the particle. Particles,microspheres, and microcapsules smaller than about 1 μm are generallyreferred to as nanoparticles, nanospheres, and nanocapsules,respectively. Capillaries have a diameter of approximately 5 μm so thatonly nanoparticles are administered intravenously. Microparticles aretypically around 100 μm in diameter and are administered subcutaneouslyor intramuscularly. See, for example, Kreuter, J., Colloidal DrugDelivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, N.Y.,pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled DrugDelivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, N.Y., pp.315-339, (1992).

Polymers can be used for ion-controlled release of the antibodycompositions disclosed herein. Various degradable and nondegradablepolymeric matrices for use in controlled drug delivery are known in theart (Langer, Accounts Chem. Res. 26:537-542, 1993). For example, theblock copolymer, polaxamer 407, exists as a viscous yet mobile liquid atlow temperatures but forms a semisolid gel at body temperature. It hasbeen shown to be an effective vehicle for formulation and sustaineddelivery of recombinant interleukin-2 and urease (Johnston et al.,Pharm. Res. 9:425-434, 1992; and Pec et al., J. Parent. Sci. Tech.44(2):58-65, 1990). Alternatively, hydroxyapatite has been used as amicrocarrier for controlled release of proteins (Ijntema et al., Int. J.Pharm. 112:215-224, 1994). In yet another aspect, liposomes are used forcontrolled release as well as drug targeting of the lipid-capsulateddrug (Betageri et al., Liposome Drug Delivery Systems, TechnomicPublishing Co., Inc., Lancaster, Pa. (1993)). Numerous additionalsystems for controlled delivery of therapeutic proteins are known (seeU.S. Pat. Nos. 5,055,303; 5,188,837; 4,235,871; 4,501,728; 4,837,028;4,957,735; 5,019,369; 5,055,303; 5,514,670; 5,413,797; 5,268,164;5,004,697; 4,902,505; 5,506,206; 5,271,961; 5,254,342 and 5,534,496).

When the composition is a liquid, the tonicity of the formulation, asmeasured with reference to the tonicity of 0.9% (w/v) physiologicalsaline solution taken as unity, is typically adjusted to a value atwhich no substantial, irreversible tissue damage will be induced at thesite of administration. Generally, the tonicity of the solution isadjusted to a value of about 0.3 to about 3.0, such as about 0.5 toabout 2.0, or about 0.8 to about 1.7.

The pharmaceutical compositions of the disclosure typically are sterileand stable under conditions of manufacture, storage and use. Sterilesolutions can be prepared by incorporating the conjugate in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated herein, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thedisclosed antigen and/or other biologically active agent into a sterilevehicle that contains a basic dispersion medium and the required otheringredients from those enumerated herein. In the case of sterilepowders, methods of preparation include vacuum drying and freeze-dryingwhich yields a powder of the disclosed antigen plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The prevention of the action of microorganisms can be accomplished byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

EXAMPLES

The following examples are provided to illustrate particular features ofcertain embodiments, but the scope of the claims should not be limitedto those features exemplified.

Example 1 Development of a Middle East Respiratory Syndrome CoronavirusVaccine

The emergence of the Middle East respiratory syndrome coronavirus as acause of severe respiratory disease ten years after the SARS-CoVoutbreak highlights the need for pre-existing platforms to facilitaterapid vaccine development for beta-coronaviruses. This exampleillustrates an MERS-CoV immunization strategy based on the MERS-CoVSpike glycoprotein (S), consisting of a full-length S DNA prime and S1subunit protein boost. The disclosed immunization strategy elicited hightiters of neutralizing antibodies against eight different MERS-CoVstrains. Vaccine-elicited murine monoclonal antibodies (mAb) werecharacterized and shown to neutralize virus by targeting the receptorbinding domain (RBD), non-RBD portions of S1, or S2. The atomicstructure of the D12 mAb in complex with RBD supported escape mutationdata that indicated several mechanisms by which antibodies block bindingto the MERS-CoV receptor, DPP4. Accordingly, gene-based priming withfull-length S and S1 protein subunit boosting induced antibodies withdiverse mechanisms of neutralization.

INTRODUCTION

Middle East respiratory syndrome coronavirus (MERS-CoV) has emerged as ahighly fatal cause of severe acute respiratory infection. Since April2012, between 1018 and 1034 cases and 366 to 409 deaths have beenattributed to the novel beta coronavirus. As human-to-human transmissionof the virus is not sustained, a large zoonotic reservoir may serve as aprincipal source for transmission events (Assiri et al., Lancetinfectious diseases 13, 752, 2013; Breban, Lancet 382, 694, 2013;Cauchemez et al., Lancet infectious diseases 14, 50, 2014; Memish etal., Emerging infectious diseases 20, 1012, 2014). The high casefatality rate, vaguely defined epidemiology, and absence of prophylacticor therapeutic measures against this novel virus have created an urgentneed for an effective vaccine, should the outbreak expand to pandemicproportions.

Past efforts to develop coronavirus vaccines have used whole-inactivatedvirus, live-attenuated virus, recombinant protein subunit, or geneticapproaches (Graham et al., Nature reviews. Microbiology 11, 836, 2013).The primary target for neutralizing antibodies is the Spike (S)glycoprotein, cleaved into two subunits: S1, which is distal to thevirus membrane; and S2, which contains both a transmembrane domain andtwo heptad-repeat sequences typical of class I fusion glycoproteins((Cavanagh, J general virology 64 (Pt 12), 2577, 1983; Buchholz et al.,PNAS 101, 9804, 2004). The S1 subunit has been the focus of mostimmunization strategies against MERS-CoV (Coleman et al., Vaccine 32,3169, 2014; Du et al., PloS one 8, e81587, 2013; Ma et al., Vaccine 32,2100, 2014), as it contains the receptor-binding domain (RBD) thatmediates virus attachment to its host receptor, dipeptidyl peptidase-4(DPP4) (Mou et al., J virology 87, 9379, 2013). Expressing the RBD onmultiple vaccine platforms can elicit neutralizing antibodies of highpotency (Du et al., J virology 88, 7045, 2014; Ohnuma et al., J virology87, 13892, 2013; Ying et al., J virology 88, 7796, 2014; Jiang et al.,Science translational medicine 6, 234ra59, 2014; Briese et al., mBio 5,e01146, 2014) that prevent viral attachment across viral strains butwill not elicit antibodies that contribute to neutralization bymediating fusion inhibition. An alternative vaccine regimen is disclosedherein, based on full-length S DNA and a truncated S1 subunitglycoprotein, to elicit a broad repertoire of antibodies with diversemechanisms of viral neutralization.

Results

Construction and Characterization of Vaccines Based on SpikeGlycoprotein.

Five vaccine constructs were designed based on sequences from theMERS-CoV S glycoprotein (FIG. 1A). The England1 strain (GenBank ID:AFY13307) was chosen based on the availability of its sequence and itsproximity to a consensus among published sequences, particularly withinthe RBD. Three plasmid vaccines were constructed that encoded: 1)full-length, membrane-anchored S; 2) transmembrane-deleted (ΔTM) Scontaining the entire ectodomain; and 3) S1 subunit only. All threeplasmids were delivered intramuscularly by needle and syringe, followedby electroporation. The two protein subunit vaccines included S-ΔTM andS1 and were delivered intramuscularly by needle and syringe with Ribiadjuvant. These five candidate vaccines were systematically evaluated inmice according to eight immunization regimens (FIG. 1B). To test theimmunogenicity of the vaccine candidates against multiple MERS-CoVstrains—without the requirement of a biosafety level 3 facility—apseudotyped reporter virus neutralization assay was developed, similarto that previously developed for SARS-CoV (Martin et al., Vaccine 26,6338, 2008; Yang et al., Nature 428, 561, 2004; Naldini et al., PNAS 93,11382, 1996; Yang et al., PNAS 102, 797, 2005). It was confirmed thatthe assay measured viral entry via the MERS-CoV receptor, DPP4, bydemonstrating that HEK 293 cells required DPP4 expression on theirsurface for efficient infection and that soluble DPP4 or anti-DPP4antibody prevented infection (FIG. 6A-D).

Full-Length S DNA and S1 Protein are the Most Immunogenic Vaccines inMice.

Mice primed either once with S1 protein or twice with S DNA and thenboosted once with S1 protein generated the highest neutralizing antibodytiters among all groups (FIG. 1B). The full-length S DNA regimen induceda significantly higher antibody response than the truncated S-ΔTM or S1DNA regimens. Antibody titers tended to be either low or undetectableafter the first dose of DNA but boosted ten-fold after immunization withS1 protein. Two doses of S1 protein achieved an IC₉₀ near that of theDNA/protein regimen. Three of the eight vaccine regimens—(1) S DNA, (2)S DNA/S1 protein, and (3) S1 protein alone—were carried forward fordetailed evaluation. S-ΔTM gave low production yields from transfectedHEK 293 cells, and was not evaluated further. Immune sera (5 weeks afterfinal boost) from these three regimens were tested against a panel ofeight pseudoviruses and found to generate equally robust neutralizingantibody titers against all strains (FIG. 1C). The sequence homologyacross strains (FIG. 7) likely accounted for the breadth ofneutralization. SARS-CoV pseudovirus was not neutralized by sera fromany of the immunized mice. Pseudovirus neutralization results werecompared with a live virus microneutralization assay for the JordanN3strain (GenBank ID: KC776174.1). The pseudovirus neutralization assaywas about ten-fold more sensitive to neutralization than the live virus,although the two assays correlated well with one another based onrelative magnitude (FIG. 8).

S DNA Prime and S1 Protein Boost Elicit Neutralizing Antibodies Directedat Antigenic Sites on Different Domains of the S Glycoprotein.

The specificity of neutralizing antibody responses against the differentsubunits of the MERS-CoV S glycoprotein were analyzed by severalmethods. First, sera were adsorbed with HEK 293T cells expressingtransmembrane-anchored versions of S, S1, S2, and RBD (FIGS. 1A and 9A)and screened for neutralization (FIG. 2A). Sera adsorbed withuntransfected cells retained 95% of their original neutralizationactivity while negative control pre-immune sera showed no capacity forvirus neutralization. The full-length S-transfected cells adsorbedvirtually all neutralization activity from the immune sera of all threevaccine groups while the RBD expressed by itself on the cell membraneonly adsorbed about half of the neutralizing activity, and only in theS1 protein group, suggesting the possibility that the conformation ofRBD presented in the full-length S and in the truncated versions maydiffer. Adsorption with S1 also removed all neutralization activity inthe S1 protein group and about 60% of neutralizing capacity in the twoDNA-primed groups. In contrast, S2 did not deplete neutralizationactivity in any of the groups, except slightly in mice vaccinated withDNA alone. It is likely that expression of S2 by itself on cellsresulted in rearrangement to the post-fusion conformation, and thereforeneutralizing epitopes present on the pre-fusion structure would not havebeen available for adsorption. Flow cytometric analysis of serum bindingto cells that expressed different S subunits gave consistent results(FIG. 9C).

Protein competition neutralization assays also recapitulated thefindings of the S-transfected cell adsorption assays (FIG. 2B).Competition with >2 μg/ml soluble RBD (FIG. 9B) reduced immune seraneutralization activity by approximately 50-60% in the S1 proteinvaccination group. As seen in the cell adsorption assay, soluble S1protein removed neutralizing activity of sera from all three vaccinegroups, though to a higher degree in the mice immunized only with S1protein compared to the groups primed with S DNA. Soluble S2 (FIG. 9B)had no impact on serum neutralization capacity and full-length S was notused as a competitor as it could not be expressed in soluble form.Overall, these data indicated that the serum neutralization activity inthe protein-only immunization group was directed primarily against theRBD. The S DNA-only or S DNA/S1 protein-induced immune sera were morecomplex as more than half of the neutralizing activity was directedagainst S1, but there was residual activity that was not absorbed orcompeted by S1. It is not conclusively known why S2 did not adsorb orcompete with these sera, but it is likely that the conformation of theexpressed S2 protein was postfusion as stated above.

DNA and Protein Vaccine Regimens Elicited Antibodies of Different IgGSubclasses.

Sera were also analyzed for the quality of the humoral response inducedby the different immunization regimens and found to elicit different IgGsubclass response patterns (FIGS. 5A and 5B). S DNA immunization inducedan IgG₂a-dominant response (geometric mean titer (GMT) IgG₂a/IgG₁ ratioof 6) while S1 protein immunization generated an IgG₁-dominant response(GMT IgG₂a/IgG₁ ratio of 0.06). The IgG subclass response pattern wasdetermined by priming immunization, based on the observation that S DNAprime/S1 protein boost regimen induced a similar pattern and magnitudeof IgG₂a polarization as the S DNA-only regimen. The IgG₂a and IgG₁subclass responses respectively reflect Th1- and Th2-biased immuneresponse patterns in mice (Stevens et al., Nature 334, 255, 1988). TheIgG₂a or Th1-biased response elicited by the DNA-containing regimens islikely due to the induction of IFN-γ producing CD8 T cells that modulateCD4 T cell differentiation (Davis et al., Human gene therapy 6, 1447,1995; Shedlock et al., J leukocyte biology 68, 793, 2000). Surprisingly,monophosphoryl lipid A-based Ribi adjuvant was not sufficient toinfluence the IgG subclass polarization toward a Th1 phenotype as wouldbe expected (Cargnelutti et al., The new microbiologica 36, 145, 2013;Chaitra et al., Vaccine 25, 7168, 2007). Thus, compared to priming witha protein, DNA priming generated a T helper response that is generallyassociated with more effective control of viral infections.

Mouse Monoclonal Antibody Functional Characterization.

The humoral response to the S DNA/S1 protein vaccine was furtherinvestigated by isolating and characterizing monoclonal antibodies(mAbs) of different specificities. Hybridomas were generated from SDNA-primed and S1 protein-boosted mice and screened for binding to theS1, RBD, and S2 domains. The final round of screens generated 45subclones (FIG. 11), four of which (D12, F11, G2, G4) were selected foradditional characterization based on their binding specificity andneutralization potency (FIGS. 3A-3B and 12). All four mAbs demonstrated,by immunofluorescence, binding to live virus (EMC strain) infected cells(FIG. 13). D12 and F11 bound the S1 subunit within the RBD. mAb G2 wasalso specific for S1, but it bound outside the RBD. G4 only bound the S2subunit (FIG. 3A). Although not as potent as the RBD-specific mAbs (FIG.3B), G2 and G4 were unique as compared to other reported mAbs againstMERS-CoV (Du et al., J virology 88, 7045, 2014; Ohnuma et al., Jvirology 87, 13892, 2013; Ying et al., J virology 88, 7796, 2014; Jianget al., Science translational medicine 6, 234ra59, 2014) in theirability to neutralize virus despite targeting epitopes outside the RBD.Soluble protein competition neutralization results were consistent withthose of the binding assays (FIG. 14A). D12 and F11 neutralization werediminished in the presence of RBD and S1, while G2 was competed by S1,and G4 neutralizing activity was only abrogated by high concentrationsof S2.

Although the two most potent neutralizing mAbs—D12 and F11—targeted theRBD, their neutralization profiles were different, when mAbneutralization capacity was tested against the panel of eightpseudotyped reporter viruses (FIG. 1C). Notably, F11 was unable toneutralize the Bisha1 strain (GenBank ID: KF600620.1) of MERS-CoV (FIG.14B), which differs from other strains by an aspartic acid to glycinesubstitution at residue 509, rendering it resistant to F11 butsusceptible to D12 neutralization. This finding was recapitulated in apseudotyped virus neutralization assay where F11 neutralization activityagainst wild-type England1 was ablated with the introduction of a D509Gmutation (FIG. 14B). D12, in contrast, neutralized both virusesirrespective of the amino acid change at position 509. In addition, theRBD 509G mutation abrogated F11 binding by ELISA but did not affect D12binding (FIG. 4E).

Structural and Mutational Analysis of the D12 Antibody in Contact with SRBD.

To provide an atomic-level understanding of neutralizing activity, theD12 antibody was crystallized as an antigen-binding fragment (Fab) incomplex with the England1 RBD (FIG. 20, FIG. 4A-4B). Additionally, theunbound structure of England 1 RBD was solved (FIG. 4C) and found to benearly identical to the RBD of the EMC strain (GenBank ID: JX869059.2),either by itself or in complex with DPP4 (Lu et al., Nature 500,227-231, 2013), as it has only one amino acid difference (F506L) in theRBD (FIG. 7). The D12 antibody forms direct contacts with the receptorbinding motif (RBM) of the RBD and the heavy chain overlaps with thecontact region between MERS RBD and human DPP4 as defined by Lu andcolleagues (Lu et al., Nature 500, 227-231, 2013). RBD residues W535 andE536, which bind to the conserved glycan on DPP4 are bound by the CDR H2and CDR H3 within the D12 paratope (FIG. 4C). Mutation of both residuesabrogated the ability of D12 to bind (FIG. 4D-4E) and neutralize virus(FIG. 14C). Additional mutational escape analysis demonstrated thatanother residue within the epitope was critical for D12 neutralizationactivity. The EMC strain of MERS-CoV escaped neutralization by D12 whenthe serine at position 532 was mutated to either a proline ortryptophan. The introduction of a proline at position 531 removes twohydrogen bonds to the D12 antibody (FIGS. 4C and 21), while successiveprolines at 531 and 532 (proline 532 is native to the RBD) is predictedto alter the side-chain orientation of adjacent residues, likelyaffecting W535 and E536 interactions with D12. Mutation to a bulkytryptophan side chain likely causes a direct clash with the CDR L3, thusprecluding binding of D12 (FIG. 4C-4E). Although it also targets theRBD, F11 neutralizes MERS-CoV by a different mechanism than D12.Site-directed mutagenesis and competition binding studies indicate thatF11 makes contact with RBD on the opposing side of D12's binding site,at and around residue 509. Binding studies also show that both D12 andF11 can bind to the RBD at the same time (FIG. 15A-15C), suggesting thepotential for additive effects on neutralization. The differing pointsof contact made by F11 and D12 on the MERS-CoV RBD are analogous to thatof other mAbs specific for the SARS-CoV RBD (Prabakaran et al., J BiolChem 281, 15829, 2006; Zhu et al., PNAS 104, 12123, 2007), suggestingconvergent mechanisms of neutralization. Furthermore, these dataelucidate mechanisms for virus neutralization that involve thedisruption of binding between the N-terminal end of the MERS-CoV RBD andits host receptor DPP4.

Full-Length S DNA or S1 Protein Prime and S1 Protein Boost Elicit Potentand Durable Neutralizing Antibody Titers in Non-Human Primates.

Of the eight vaccine regimens tested in mice, the three most immunogenicwere taken forward for evaluation in NHPs (FIG. 5A). Comparable to themice, NHPs primed with either S1 protein (plus aluminum phosphateadjuvant) or S DNA (followed by electroporation) and boosted with S1protein (plus aluminum phosphate adjuvant) generated the highestneutralizing antibody titers, as measured by the pseudotyped virusneutralization assay, compared to the S DNA-only group (FIG. 5B). Bothgroups initially had low antibody titers after priming that increased 10to 100-fold after boosting. IC₉₀ values after the final boost wereapproximately 1 log₁₀ higher in mice than NHPs, which could be due tothe different animal models, vaccine doses or adjuvants used in the twostudies. Antibody titers remained high, however, at more than 2.5 log₁₀at 10-weeks post-boost and persisted at higher levels in the DNA-proteingroup. A microneutralization assay with the MERS-CoV JordanN3 strain wascompared with pseudotyped neutralization assay and demonstrated similarresults. Sera from the two groups immunized with S DNA bound allepitopes recognized by the four previously characterized murineantibodies (FIG. 17). Sera from NHPs immunized with S1 protein alone,however, blocked mAbs targeted to the RBD (D12, F11) and non-RBD S1subunit (G2) but not the S2 subunit (G4).

Full-Length S DNA or S1 Protein Prime and S1 Protein Boost ConferProtection to Non-Human Primates Challenged with MERS-CoV.

Immunized NHPs were challenged with the JordanN3 strain of MERS-CoV at19 weeks post-boost and demonstrated earlier and diminished peak lunginfiltrates compared to unvaccinated NHPs (FIG. 5C). Immunization withS1/S1 protein or S DNA/S1 protein resulted in a respective four- tosix-fold reduction in the peak proportional volume of pulmonaryconsolidation (FIG. 5C), as demonstrated by high resolution computedtomography (FIG. 5D and FIGS. 18A-18C) and analyzed by a previouslydescribed method of lung segmentation (Mansoor et al., IEEE transactionson medical imaging, 33, 2293-2310, 2014). NHPs immunized with S DNA/S1protein experienced a lower peak volume of pulmonary disease than theS1/S1 protein group and cleared the pulmonary infiltrates more rapidly.All three groups exhibited a boost in anti-S1 IgG antibody and virusneutralization titers after challenge. A greater magnitude of rise intiters was observed in the unvaccinated group. Although virus isolatescould not be consistently obtained from oral, nasopharyngeal, ortracheal swabs in any of the three challenged groups, anti-S1 IgG andneutralizing antibodies were detected two weeks after challenge in theunvaccinated group and boosted in the vaccinated groups (FIGS. 19A-19B),suggesting viral antigen was produced by infection. No pre- orpost-challenge differences between vaccinated and unvaccinated NHPs wereobserved in clinical symptoms, laboratory values, or histopathologyparameters. Additionally, there was no statistically significantcorrelation between neutralization antibodies either at day of challengeor at peak (two weeks after the last boost) with the percentage ofabnormal lung volume (FIGS. 19C-19D).

Discussion

In summary, DNA expression vectors and soluble protein immunogens weredeveloped to elicit cross-reactive neutralizing antibodies againstknown, circulating strains of MERS-CoV. The induced neutralizingantibodies targeted several regions of the MERS-CoV Spike protein.Immunization with DNA expressing full-length S followed by S1 subunitprotein yielded potent neutralizing mAbs in both mice and NHPs. In micethose mAbs were cross-reactive against multiple MERS-CoV strains anddirected against the RBD of the S glycoprotein-preventing attachment—andagainst epitopes outside the RBD in the S1 and S2 subunits. Further, thecurrent immunization strategy is the first to induce MERS-CoVneutralizing antibodies that target multiple epitopes, both within andoutside the RBD, which may potentially improve immunogenicity and reducethe likelihood of escape mutations.

Co-crystal structures and analysis of escape mutations provided amechanistic explanation for neutralization by the RBD-directedneutralizing antibodies. The RBD-specific antibodies we isolated arecomparable to others reported in the literature, particularly thoseisolated from human antibody phage libraries (Tang et al., PNAS, 111,E2018-2026, 2014), however D12 and F11 target slightly differentepitopes and are approximately a 1000-fold more potent in theirneutralization capacities. D12 appears to be highly novel in recognitionwhile F11 does show some epitope overlap with the mAb 3B12 in the T512loop region but the added sensitivity of 3B12 to mutations at positions540 and 542 is not seen with F11, thus highlighting the difference intheir respective epitopes.

Compared to protein alone, S DNA prime/S1 protein boost immunizationyielded a more functionally diverse repertoire of neutralizingantibodies and also generated a Th1-biased immune response. The DNAprimed regimen also offered greater and earlier protection in challengedNHPs, suggesting the activation of effector CD8+ T cells. Thus, while aprotein-only MERS vaccine may be the simpler approach, there arepotential advantages to inclusion of a DNA prime. DNA priming mayimprove the durability of the immune response (Caulfield et al., Jvirology 76, 10038, 2002) and, through modifications in the boostinterval, might improve the magnitude and functional properties of theantibody response as it does for conventional influenza vaccines(Ledgerwood et al., Lancet infectious diseases 11, 916, 2011; Ledgerwoodet al., J infectious diseases 208, 418, 2013); Khurana et al., J ofinfectious diseases 208, 413, 2013)).

The presentation of the S trimer in a native conformation on the surfaceafter DNA immunization may have contributed to the generation of adiverse set of antibodies that could neutralize MERS-CoV by targetingepitopes outside the RBD. In addition to providing a broader array offunctional antibody responses, non-RBD antibodies may aid in solving thetrimeric S glycoprotein structure by targeting quarternary epitopes andstabilizing the prefusion conformation.

Materials and Methods

Ethics Statement.

Animal experiments were carried out in compliance with all pertinentU.S. National Institutes of Health regulations and policies.

DNA and Protein Vector Constructs.

To evaluate which vaccine candidates and immunization regimens couldgenerate a potent neutralizing antibody response, DNA vaccines ofMERS-CoV England1 strain S and two truncated versions, S-ΔTM and S1 wereconstructed (FIG. 1A). Protein subunit vaccines for S-ΔTM and S1 werealso constructed (FIG. 1A). The MERS-CoV S gene (strain England1,GenBank ID: AFY13307) was synthesized according to a previouslydescribed method (Yang et al., J Virol., 78, 5642-5650, 2004). Briefly,amino acid sequences were obtained from GenBank™, reverse-translated andcodon-optimized for human cell expression. 75-base pair oligonucleotidesets with 25-base pair overlaps were then synthesized and gel purified.Oligonucleotides were assembled into DNA fragments using the Pfu TurboHotstart DNA polymerase (Stratagene, La Jolla, Calif.) at a 50° C.-65°C. gradient annealing temperature. DNA fragments were cloned into thepCR-Blunt II-Topo vector (Invitrogen, Carlsbad, Calif.) and sequenced.Fully corrected DNA fragments for each gene were ultimately cloned intothe mammalian expression vector VRC8400 (Barouch et al., J virology 79,8828, 2005; Catanzaro et al., Vaccine 25, 4085, 2007).

All other truncated versions and domain-swapping mutants weresynthesized by PCR, using full length S as the template (FIG. 1A). ThePCR fragments were digested with Xbal and BamHI and cloned into theVRC8400. All mutations and fused constructs were confirmed bysequencing. Proteins were expressed in the Expi293 cell line bytransfection with expression vectors encoding corresponding genes. Thetransfected cell culture supernatants were collected and purifiedthrough a HisTrap HP column and a Hiload 16/60 Superdex column (GEhealthcare, Piscataway N.J.) according to manufacturer's instructions.

In accordance with manufacturer protocols, cDNAs were synthesizedencoding S using the QuikChange XL kit (Stratagene, La Jolla, Calif.)and introduced divergent amino acids into the parental S strain(England 1) predicted from the translated sequences of other strains(strain Batin1, GenBank ID KF600628), (strain Bisha1, GenBank ID:KF600620), (strain Buraidah1, GenBank ID: KF600630), (strain EMC,GenBank ID: AFS88936), (strain Hasa14b, GenBank ID: KF600643), (strainJordanN3, GenBank ID: KC776174), (strain Munich, GenBank ID: KF192507).All constructs were confirmed by sequencing.

Cell Lines and Pseudovirus Production.

293, 293T (ATCC) and Huh7.5 cells were cultured in DMEM supplementedwith 10% fetal bovine serum, 2 mM glutamine and 1×penicillin/streptomycin in a 37° C. incubator containing 5% CO2. Toproduce pseudovirus, 15×10⁶293 T cells were aliquoted into a 15 cm plateand co-transfected with three plasmids (17.5 μg of packaging plasmidpCMVAR8.2, 17.5 μg of transducing plasmid pHR′ CMV-Luc, and 1 μg ofCMV/R-MERS-CoV S plasmid) and calcium phosphate reagent (Invitrogen,Carlsbad Calif.) as described previously (Naldini et al., PNAS 93,11382, 1996; Yang et al., PNAS 102, 797, 2005). After overnightincubation, media was replaced with fresh 10% FBS-DMEM. 48 hours later,the pseudovirus-containing supernatants were collected, filtered througha 0.45 μm filter, aliquoted, and frozen at −80° C. Pseudovirus wastitrated by first plating 1×10⁴ Huh7.5 cells per well in a 96-wellwhite/black Isoplate (PerkinElmer, Waltham, Mass.) and culturingovernight. Media was removed and 2-fold serial dilutions of pseudoviruswere added to the cells. After 2 hours of incubation, 100 μl of freshmedia was added. Cells were lysed 72 hours later and 50 μl of luciferasesubstrate (Promega, Madison, Wis.) was added to each well. Luciferaseactivity was measured according to relative luciferase unit (CPS) by theMicrobeta luminescence counter (PerkinElmer, Waltham, Mass.).

Phylogenetic Analysis.

Full-length amino acid sequences of S from MERS-CoV strains England1,Munich, EMC, Buraidah1, Bisha1, Batin1, Hasa14b and Jordan N3 werealigned using MAFFT L-INS-i (version 6.8.6.4, trex.uqam.ca). Aphylogenetic tree was generated by the average linkage (UPGMA) method.The phylogenetic tree was drawn on iTOL server (itol.embl.de).

Mouse Immunizations. Female BALB/cJ mice at age 6-8 weeks (JacksonLaboratory, Bar Harbor, Me.) were immunized according to severaldifferent regimens that fall within three categories: (i) 3×S DNA, (ii)2×S DNA-S1 protein and (iii) 2×S1 protein. Within the first DNA-onlycategory (i) three groups of mice were injected with plasmid DNAencoding either MERS-CoV full-length S (group 1), S with a deletedtransmembrane unit (S-ΔTM) (group 2) or S1 (group 3). Injections weregiven intramuscularly and followed by electroporation with AgilePulseSystem (Harvard Apparatus, Holliston, Mass.) at weeks 0, 3 and 6. Theheterologous regimen DNA-protein groups (ii) were injected twice withplasmid DNA either encoding MERS-CoV full-length S (group 4), S-ΔTM(group 5), or S1 (group 6) at weeks 0 and 3 as described and boostedwith either MERS-CoV S-ΔTM (group 5) or S1 (groups 4 and 6) protein plusRibi adjuvant (Sigma-Aldrich, St. Louis, Mo.) at week 6. The homologousprotein groups (iii) were injected twice with MERS-CoV S-ΔTM (group 7)or S1 protein (group 8) plus Ribi adjuvant at weeks 0 and 4.

DNA immunizations were given as bilateral quadriceps muscle injectionsof 20 μg of plasmid DNA in a total volume of 100 μl of PBS (50 μl eachside). Protein immunizations were given as bilateral quadricepsinjections of 10 μg of protein in 50 μl PBS mixed with an equal volumeof Ribi adjuvant in a total volume of 100 μl (50 μl each side). Twoweeks after each injection, sera were collected for measurement ofantibody responses.

Non-Human Primate (NHP) Immunizations.

Eighteen (6 female and 12 male) Indian rhesus macaques (Macaca mulatta)weighing 3.2-4.8 kg and with a mean age of 4.4 years were randomlyassigned to three groups according to sex and body weight and immunizedaccording to one of three different vaccine regimens: 3×S DNA, 2×SDNA-S1 protein and 2×S1 protein. Sample size was based on conventionwithin the literature. Within the DNA-only group, six NHPs were injectedwith plasmid DNA encoding MERS-CoV full-length S. Injections were givenintramuscularly and followed by electroporation(manufacturer-recommended setting) with AgilePulse System (HarvardApparatus, Holliston, Mass.) at weeks 0, 4 and 8. Six NHPs in the SDNA-S1 protein group were injected with plasmid DNA encoding MERS-CoVfull-length S at weeks 0 and 4 and boosted with MERS-CoV S1 protein andaluminum phosphate adjuvant (Brenntag Biosector, Frederikssund, Denmark)at week 8. Six NHPs in the protein-only group were injected with 100 μgof MERS-CoV S1 protein and aluminum phosphate adjuvant at weeks 0 and 8.DNA immunizations were given as bilateral quadriceps and biceps muscleinjections of 1 mg of plasmid DNA in a total volume of 1000 μl of PBS(250 μl each site). Protein immunizations were administered as bilateralquadriceps and biceps injections of 100 μg of protein in 500 μl PBSmixed with an equal volume of aluminum phosphate adjuvant in a totalvolume of 1000 μl (250 μl each site). Sera were collected formeasurement of antibody responses two weeks after each injection andevery two-to-four weeks thereafter through week 18.

Non-Human Primate (NHP) Virus Challenge.

Eighteen Indian rhesus macaques-6 unvaccinated, 6 vaccinated with SDNA/S1 protein, 6 vaccinated with S1/S1 protein—were included in a viruschallenge experiment with the Jordan N3 strain of MERS-CoV (GenBank ID:KC776174.1) in an approved high containment facility. Due to highintensity of the imaging studies conducted, NHPs were divided intoseparate challenge cohorts. Prior to challenge, stock virus was dilutedto a target concentration of 5×10⁶ PFU/mL. Approximately 19 weeksfollowing their last vaccine boost, NHPs were inoculated with 1 mL ofchallenge inoculum by intra-tracheal administration using a laryngoscopeand lubricated catheter. Following challenge, virus challenge inoculumwas assayed to determine actual delivered dose. Delivered doses were3.1×10⁶, 3.6×10⁶ and 3.4×10⁶ PFU for Cohorts 1, 2 and 3, respectively.Following virus challenge, all NHPs in each of the three groups weremonitored and assessed by clinical, laboratory, and radiologicparameters at days 3, 6, 9, and 14 post-challenge. On day 28post-challenge all NHPs underwent necropsy.

Non-Human Primate Radiologic Assessment.

Prior to and following virus challenge, NHPs in each of the three groupsunderwent chest imaging by computed tomography, using the PET/CT GeminiTOF imager (Philips, Andover, Mass.). At the conclusion of dataacquisition, images were evaluated and analyzed by two board-certifiedradiologists. Lung pathology was quantitatively assessed longitudinallyusing a previously described pulmonary imaging analysis technique (Zhuet al., PNAS, 104, 12123-12128, 2007). Briefly, an automated imagedelineation algorithm was used to determine the lung region of interest(ROI). This procedure was followed by a machine learning algorithm,named “random forest”, that assesses the tissue class (i.e., normal orabnormal) of every CT image pixel within the ROI. Once machine thelearning algorithm is finalized, the total lung capacity, pathologyvolume (if present) and their proportion (pathology volume/total lungvolume) are calculated as quantitative metrics for describing diseaseseverity. Additionally, we developed a three-dimensional visualizationof the lung pathology based on the results of this algorithm. Inprevious experiments, ground glass opacities (GGO) and consolidationshave mostly constituted the abnormal imaging patterns on CT scans.Therefore, the machine-learning algorithm was optimized according tothese patterns. Moreover, an additional identification step for airwayand vessel recognition was included to minimize false positive findingsand increase the efficiency of the computer-aided detection system.

Pseudovirus Neutralization Assay.

Huh7.5 cells (10,000 cells per well) were plated into 96-wellwhite/black Isoplates (PerkinElmer, Waltham, Mass.) the day beforeinfection. Serial dilutions of serum were mixed with different strainsof titrated pseudovirus (target CPS of 200,000 in a linear titrationrange), incubated for 30 minute at room temperature and added to Huh7.5cells in triplicate. Following 2 hours of incubation, wells werereplenished with 100 μl of fresh media. Cells were lysed 72 hours laterand 50 μl of luciferase substrate (Promega, Madison, Wis.) was added toeach well. Luciferase activity was measured according to relativeluciferase unit (CPS) by the Microbeta luminescence counter(PerkinElmer, Waltham, Mass.). IC₉₀ neutralization titers werecalculated for each individual mouse serum sample.

Microneutralization Assay.

Two-fold dilutions of heat-inactivated, grouped sera were tested in amicroneutralization assay for the presence of antibodies thatneutralized the infectivity of 100×TCID50 of MERS-CoV JordanN3 strain inVero cell monolayers, using four wells per dilution in a 96-well plate.Viral cytopathic effect (CPE) was read on days 3 and 4. The dilution ofserum that completely prevented CPE in 50% of the wells was calculatedby the Reed-Muench formula.

Cell Adsorption and Protein Competition Neutralization Assays.

Mouse sera from mice immunized with 3×full-length S DNA, 2×full-length SDNA+S1 protein, and 2×S1 protein were diluted to concentrations thatyielded 90-95% of neutralization activity in Huh7.5 cells (1:400 for3×DNA and pre-immunization sera, 1:4000 for 2×DNA-protein and 2xproteinsera). The diluted sera were then incubated with 293 cells transfectedwith MERS-CoV S, RBD-HA™, S1-TM, S2-TM for 1 hour at 4° C. on a rocker.After the cells were spun down, supernatants were collected forneutralization assays. To test whether adsorption was complete, residualsera were used to stain against MERS-CoV S, RBD-HA™, S1-TM,S2-TM-transfected 293 cells. Pre-immune sera and untransfected cellswere included as negative controls.

The protein competition assay included soluble MERS-CoV S1, RBD and S2that were diluted and incubated for 1 hour at 37° C. with mouse sera ormAbs diluted to yield a 90-95% neutralization activity. Sera/proteinmixtures were then incubated with titrated pseudovirus for 30 minutes atroom temperature. The final mixture of protein, serum and pseudoviruswas added to 96-well white/black Isoplate (PerkinElmer, Waltham, Mass.)of cells plated the day before. After 2 hours of incubation, 100 μl offresh media was added to each well. Cells were then lysed 72 hours laterand 50 μl of luciferase substrate (Promega, Madison, Wis.) was added toeach well. Luciferase activity was measured according to relativeluciferase unit (CPS) by the Microbeta luminescence counter(PerkinElmer, Waltham, Mass.).

Cell Surface Binding by FACS.

293 cells were transfected with plasmids expressing MERS-CoV S, RBD-HA™,S1-TM, and S2-TM. 24 hours later, cells were detached with 4 mM EDTA inPBS and stained with Vivid® viability dye (Invitrogen, Carlsbad,Calif.). Cells were then stained with sera (1:200 dilution) from miceimmunized with 3×full-length S DNA, with 2×full-length S DNA+S1 proteinand 2×S1 protein. Cells were subsequently stained with anti-mouse-PE(Santa Cruz Biotechnology, Santa Cruz, Calif.) and sorted with an LSR(BD Biosciences, San Jose, Calif.). Data were analyzed by FlowJosoftware (Tree Star, Inc, Ashland, Oreg.).

Competition ELISA.

The detailed methods followed for competition ELISA assays have beenpublished elsewhere (Tomaras et al., PNAS, 110, 9019-9024, 2013; Kong etal., J. Virol., 86, 12115-12128, 2012). Briefly, mAbs F11, D12, G2 andG4 were biotinylated with EZ-Link Sulfo-NHS-Biotinylation Kit (ThermoFisher Scientific, Waltham, Calif.) and titrated on MERS-CoV S1 coatedplates. Avidin D HRP conjugate (Vector Laboratories, Burlingame Calif.)and TMB (KPL, Gaithersburg Md.) were used to allow color development.OD450 nm was detected with SpectraMax Plus (Molecular Devices, SunnyvaleCalif.). The concentration of biotin-mAb in the linear range of thetitration curve was chosen for competition ELISA. The NHP sera fromthree vaccinated groups were serially diluted into the biotin-mAb withthe designed concentration and added into S1-coated plates. Biotin-mAbalone was used as the binding control. The percent inhibition by the NHPsera was calculated as follows: 100-(NHP sera+biotin-mAbreading)/biotin-mAb reading)×100.

Microscopy.

WHO-Vero cells grown to 60% confluence on 12 millimeter glass coverslipswere infected with MERS-CoV (EMC strain) at an MOI of 1 PFU/cell. At 24hours post-infection, medium was aspirated, and cells were fixed andpermeabilized in methanol at −20° C. overnight. Viral studies withMERS-CoV were performed in a BSL-3 laboratory using protocols reviewedand approved by the Institutional Biosafety Committee of VanderbiltUniversity and the Centers for Disease Control for the safe study andmaintenance of MERS-CoV. Cells were rehydrated in PBS for 20 min andblocked in PBS containing 5% bovine serum albumin (BSA). Blockingsolution was aspirated, and cells were washed with immunofluorescence(IF) assay wash solution (PBS containing 1% BSA and 0.05% Nonidet P-40)at room temperature. Cells were washed in IF wash solution 3 times for 5min per wash and incubated in secondary antibodies (Goatα-mouse-AlexaFluors 546 (1:1500), Invitrogen Molecular Probes) for 30min. Cells were washed 3 times for 5 min per wash, followed by a finalwash in PBS, and then rinsed in distilled water. Coverslips were mountedwith Aquapolymount (Polysciences) and visualized by immunofluorescencemicroscopy on a Nikon Eclipse TE-20005 wide field fluorescentmicroscope. Cells were imaged using a 40× oil-immersion lens throughDIC, Cy3, and DAPI filters. Resulting images were merged and assembledusing Nikon Elements, ImageJ, and Adobe Photoshop CS2. Infected andmock-infected cells were processed in parallel.

Binding Studies of Vaccine-Induced Mouse Monoclonal Antibodies toMERS-CoV Antigens Using Biolayer Interferometry.

A fortéBio Octet Red384 instrument was used to measure binding kineticsof RBD, S1, and S2 MERS-CoV molecules to the neutralizing antibodies(F11, D12, G2, and G4). All the assays were performed with agitation setto 1,000 rpm in phosphate-buffered saline (PBS) buffer supplemented with1% bovine serum albumin (BSA) in order to minimize nonspecificinteractions. The final volume for all the solutions was 40-50 μl/well.Assays were performed at 30° C. in solid black tilted-bottom 384-wellplates (Geiger Bio-One). Mouse antibodies (40-50 μg/ml) in PBS bufferwas used to load anti-mouse IgG Fc capture (AMC) probes for 300 s.Typical capture levels were between 1 and 1.5 nm, and variability withina row of eight tips did not exceed 0.1 nm. Biosensor tips were thenequilibrated for 180 s in PBS/1% BSA buffer prior to binding assessmentof the RBD and S1, MERS-CoV molecules in solution (250 to 7.8 nM) for300 s; binding was then allowed to dissociate for 300 s. Ahuman-Fc-MERS-CoV S2 chimeric molecule (S2-hFc) was used to loadanti-human IgG Fc capture (AHC) probes for 300 s and binding to G4 Fab(X to Y nM) was assessed as described for the other antibodies.Dissociation wells were used only once to prevent contamination.Parallel correction to subtract systematic baseline drift was carriedout by subtracting the measurements recorded for a sensor loaded withmouse monoclonal antibodies or hFc-S2 incubated in PBS/1% BSA. Dataanalysis and curve fitting were carried out using Octet software,version 8.0.

Experimental data were fitted with the binding equations describing a1:1 interaction. Global analyses of the complete data sets assumingbinding was reversible (full dissociation) were carried out usingnonlinear least-squares fitting allowing a single set of bindingparameters to be obtained simultaneously for all concentrations used ineach experiment.

Generation of Hybridomas.

After the third DNA or second protein immunization, immune sera wereassessed for S1 binding activity by ELISA and MERS-CoV pseudovirusneutralization capacity. Mice that yielded antisera with good bindingactivity and high neutralization titers were boosted with an additional20 μg of S1 delivered intramuscularly. Three days post-boost splenocyteswere harvested and fused with Sp2/0 myeloma cells (ATCC, Manassas, Va.,USA) using PEG 1450 (50% (w/v), Sigma, St. Louis, Mo., USA) according tothe standard methods. Cells were cultured and screened in RPMI completemedium that contained 20% FCS and 1×HAT (100 μM hypoxanthine, 0.4 μMaminopterin, 16 μM thymidine, Sigma). Supernatants from resultinghybridomas were screened for binding, by ELISA, to MERS-CoV S1, RBD, or5-ΔTM as well as for neutralizing activity. Subclones were generated bythe limiting dilution methods. After three rounds of screening andsubcloning, stable antibody-producing clones were isolated and adaptedto hybridoma-serum free medium (Life technologies, Grand Island, N.Y.,USA). Supernatants were collected from selected hybridoma clones andpurified through a protein A-sepharose column (GE Healthcare,Piscataway, N.J., USA). Generated monoclonal antibodies were isotypedwith the Pierce rapid isotyping kit according manufacturer'sinstructions. The DNA and protein sequences of the G2, G4, D12 and F11antibodies are provided as SEQ ID NOs: 114-129.

X-Ray Crystallography.

A construct encoding the receptor-binding domain (RBD) of MERS-CoV Eng1Spike glycoprotein spanning residues 367 to 606 with a c-terminal HRV-3ccleavage site and His₆ purification tag was produced in GnTi⁻ cells aspreviously described for 293 cell expression. Protein was purified byNiNTA affinity chromatography followed by gel filtration using 1×PBS asbuffer. Monoclonal antibodies used in crystallization studies werepurified using Protein G resin. Fabs were prepared using the PierceMouse IgG₁ Fab kit following typical protocols. Following purification,RBD England1 was concentrated to ˜5 mg/ml for crystallization trials. Toprepare RBD England1 Fab complexes for crystallization studies, the RBDmolecule was mixed with the Fab in a 1:1.5 molar ratio and allowed tosit for 30 mins at room temperature. The complexes were purified by sizeexclusion chromatography (Superdex S200; GE Healthcare) and concentratedto −5-8 mg/ml.

Crystallization screening was carried out using a Mosquitocrystallization robot, using the hanging drop vapor diffusion method at20° C. by mixing 0.1 μl of protein complex with 0.1 μl of reservoirsolution. Once initial crystal conditions were observed, furthercrystallization trials to improve crystal size and shape were carriedout by hand using 0.5 μl of protein complex with 0.5 μl of reservoirsolution.

Crystals of RBD England1 were obtained using a reservoir solution of 0.1M Tris-HCl pH 8.5, 10% MPD, 29% PEG 1,500. Crystals were cryogenicallycooled in liquid nitrogen using mother liquor containing 20% ethyleneglycol as a cryoprotectant. Crystal form 1 of the D12 Fab:RBD England1complex were obtained using a reservoir solution of 0.1 M sodium acetatepH5.5, 50 mM sodium chloride, 10% PEG 400, 11% PEG 8,000. Crystals werecryo-cooled in liquid nitrogen using mother liquor containing 22%ethylene glycol as a cryoprotectant. Crystal form 2 of D12 Fab:RBDEngland1 were obtained using a reservoir solution of 0.1 M sodiumCacodylate pH 6.5, 80 mM magnesium acetate, 14.5% PEG 8,000. Crystalswere cryo-cooled in liquid nitrogen using mother liquor containing 15%2R-3R butanediol as a cryoprotectant.

Data for all crystals were collected at a wavelength of 1.00 Å atSER-CAT beamlines ID-22 and BM-22 (Advanced Photon Source, ArgonneNational Laboratory). All diffraction data were processed with theHKL2000 suite (Otwinowski et al., Methods Enzymol. 276, 307, 1997),structures were solved by molecular replacement using PHASER (McCoy etal., J applied crystallography 40, 658, 2007), and iterative modelbuilding and refinement were performed in COOT (Emsley et al., Actacrystallographica. Section D, Biological crystallography 66, 486, 2010)and BUSTER-TNT (Blanc et al., Acta crystallographica. Section D,Biological crystallography 60, 2210, 2004), respectively. For the RBDEngland1 crystals, a molecular replacement solution with two moleculesper asymmetric unit was obtained by using the PDB ID 4KR0 (Lu et al.,Nature 500, 227, 2013) molecule B as a search model. For the two crystalforms of the D12:RBD England1 complex, a molecular replacement solutionwas obtained using the PDB ID 4KR0 molecule B as a search model for theRBD, PDB ID 1IGM (Fan et al., J mol biol 228, 188, 1992) as a searchmodel for the Fab variable domain, and the mouse constant region of theFab F26G19 PDB ID 3BGF (Pak et al., J mol biol 388, 815, 2009) as asearch model for the Fab constant domain. In both crystal forms, twoRBD-Fab complexes were obtained per asymmetric unit.

During refinement a cross validation (Rfree) test set consisting of 5%of the data was used to assess the model refinement process withstructure model validation carried out using MolProbity (Adams et al.,Acta crystallographica. Section D, Biological crystallography 66, 213,2010; Chen et al., Acta crystallographica. Section D, Biologicalcrystallography 66, 12, 2010). The RBD England1 model was refined to afinal Rfactor value of 19.5% and Rfree value of 25% with 99% residues inthe favored region of the Ramachandran plot with no outliers. The D12:RBD England1 crystal from 1 gave a structure model with a final Rfactorvalue of 17.8% and Rfree value of 23.8% with 99% residues in the favoredregion of the Ramachandran plot with no outliers. The D12: RBD England1crystal form 2 gave a structure model with a final Rfactor of 22.5% andRfree value of 26.1% with 99% residues in the favored region of theRamachandran plot.

MERS-CoV Monoclonal Antibody Escape Mutations.

Cells and viruses. WHO-Vero or Vero81 cells (Vero) were maintained inDulbecco's modified eagle's medium (Invitrogen) containing 7% FBS,supplemented with penicillin, streptomycin, and amphotericin B. MERS-CoVEMC strain generated from an infectious clone (Scobey et al., PNAS 110,16157, 2013) was propagated and assessed by plaque assay on Vero cells.All incubations of cells and virus were at 37° C. in a 5% CO2atmosphere. All viral studies were performed in certified BSL3laboratories and exclusively within biological safety cabinets usingprotocols for safe study, maintenance, and transfer of that have beenreviewed and approved by the Institutional Biosafety Committees ofVanderbilt University.

Plaque Reduction Neutralization Assay.

Starting at a concentration of 10 μg/ml, mAbs were serially diluted5-fold and mixed with an equal amount of virus a total of six times.Virus-mAb mixtures were incubated at 37° C. for 30 min, then 200 μl ofeach mixture was used to inoculate Vero cell monolayers in 6-well platesin duplicate. Following 1 h incubation, cells were overlaid withcomplete media plus 1% agar. Plaques were visualized and counted between48-52 h post-infection. The amount of infectious virus in the presenceof each mAb concentration was calculated and graphed.

Passage for Antibody Escape.

Vero cells were plated in 25-cm² flasks the day before infection.Immediately prior to infection, media was replaced with 3 ml DMEMcontaining 3.5% serum. mAb was then added to each flask and cells wereinfected with MERS-CoV at an MOI of 0.1 PFU/ml. Two days later, mAb wasadded to new flasks of Vero cells, and 10 μl of the supernatant from theprevious infection was added to the new flask. Three separate lineageswere carried in parallel. Five passages were completed, with increasingamounts of mAb at each passage. Following the fifth passage, thesupernatant was removed, aliquoted and frozen. A small sample of eachvirus was thawed and titered by plaque assay in the presence and absenceof mAb. Ten plaques from each lineage were picked from wells that weretitered in the presence of mAb. Each plaque was used to inoculate a25-cm² flasks seeded with cells the day before infection. Virus wasallowed to replicate for 2 d, then the supernatant was removed,aliquoted and frozen. Cells were lysed using TRIzol reagent (LifeTechnologies Corp.) according to manufacturer's instructions and thelysates frozen.

Sanger (Dideoxy) Sequence Analysis of the Spike Glycoprotein.

Total cellular RNA was extracted from lysates of mAb-resistant plaqueclones using TRIzol reagent. RNA, subjected to RT-PCR withSuperscriptIII (Life Technologies Corp.) and EasyA (AgilentTechnologies, Inc.), was set to the following thermal cyclingconditions: 50° C.×30 min, 95° C.×5 min, 40 cycles of 95° C.×30 sec, 45°C.×30 sec, 72° C.×1 min and 72° C.×10 min. The MERS-CoV Spike gene wasamplified in two amplicons, each about 3 kilobases in length. Eachamplicon was sequenced using either 2 or 4 primers, to give completecoverage of the gene. The resulting sequences were assembled andcompared with the expected, theoretical sequence for MERS-CoV Spike anddifferences noted.

Statistical Analysis.

Geometric means (GMT) and 95% confidence intervals (CI) were calculatedfor all antibody titers. Means and standard errors were calculated forall other data. P values were calculated with a two-tailed, unpaired,nonparametric Mann-Whitney test using Prism software (Version 6.04,GraphPad, La Jolla, Calif.). Statistically significant differences weremet a threshold alpha value of 0.05. Statistical variation within eachdataset is represented as the standard error in each of the figures.Pearson correlation coefficients and associated p values were calculatedusing Prism software. Variances were generally similar to justify theuse of nonparametric statistical tests.

Example 2 MERS-CoV S Protein Specific Antibodies

The example describes isolation and characterization of MERS-CoV Sprotein specific antibodies.

Antibody Isolation

Isolation of the G2, G4, D12, and F11 murine antibodies is describedabove. The DNA and protein sequences of the heavy and light chainvariable domains are provided as SEQ ID NOs: 114-129.

The JC57-13, JC57-11, JC57-14, FIB_B2, and FIB_H1 antibodies wereisolated from rhesus macaques vaccinated with MERS-CoV 2×DNA-protein(FIG. 5B) using single B cell sorting and IgG heavy and light chaincloning technology as described in detail previously (Wu, et al. Science2010 DOI: 10.1126/science.1187659). Briefly, MERS-CoV antigen specific Bcells were identified by staining with a live/death marker (VIVID)followed by a panel of fluorescently labeled antibodies for CD3, CD4,CD8, CD14, CD20, IgM, IgG and two probes, MERS-CoV RBD and S1 proteins.RBD+ and/or S1+ single B cell was sorted into 96-well PCR platecontaining lysis buffer. Reverse transcription (RT) reaction was carriedout to produce cDNA followed by 2-round of nested PCR to amplify the IgGheavy and the light chain genes. 2^(nd) PCR products were sequenced. PCRproducts that gave a productive IgH, Igκ or Igλ rearranged sequence werere-amplified from the 1^(st) round PCR using custom primers containingunique restriction digest sites and subsequently cloned into thecorresponding Igγ1, Igκ or Igλ expression vectors. Monoclonal IgGantibody was expressed by co-transfection of Expi-293 cells with thepaired heavy and light plasmids and purified using recombinant protein-Acolumn (GE Healthcare). The protein and nucleic acid sequences of theheavy and light chain variable regions of the JC57-13, JC57-11, JC57-14,FIB_B2, and FIB_H1 antibodies are provided as SEQ ID NOs: 1-12 and51-58. IMGT CDR sequences are provided in Table 1.

The C2 (CDC_C2), C5 (CDC_C5), A2 (CDC_A2), and A10 (CDC_A10) antibodieswere isolated from a human MERS-CoV survivor using single B cell sortingand IgG heavy and light chain cloning technology as described in detailpreviously (Wu, et al. Science 2010 DOI: 10.1126/science.1187659).Briefly, MERS-CoV antigen specific B cells were identified by stainingwith a live/death marker (VIVID) followed by a panel of fluorescentlylabeled antibodies for CD3, CD4, CD8, CD14, CD20, IgG and two probes,MERS-CoV RBD and S1 proteins. RBD+ and/or S1+ single B cell was sortedinto 96-well PCR plate containing lysis buffer. Reverse transcription(RT) reaction was carried out to produce cDNA followed by 2-round ofnested PCR to amplify the IgG heavy and the light chain genes. 2^(nd)PCR products were sequenced. PCR products that gave a productive IgH,Igκ or Igλ rearranged sequence were re-amplified from the 1′ round PCRusing custom primers containing unique restriction digest sites andsubsequently cloned into the corresponding Igγ1, Igκ or Igλ expressionvectors. Monoclonal IgG antibody was expressed by co-transfection ofExpi-293 cells with the paired heavy and light plasmids and purifiedusing recombinant protein-A column (GE Healthcare). The protein andnucleic acid sequences of the heavy and light chain variable regions ofthe C2, C5, A2, and A10 antibodies are provided as SEQ ID NOs: 35-50.IMGT CDR sequences are provided in Table 1.

Unless indicated otherwise, the heavy and light chain variable regionsof the identified antibodies were expressed with human IgG₁ constantregion (Wu, et al. Science 2010 DOI: 10.1126/science.1187659) for thecharacterization assays described below.

Antibody Characterization

Antibody specificity was detected by binding to MERS-CoV RBD, SE or S2coated ELISA plates (see FIG. 22). RBD-specific antibodies include F11,D12, JC57-11, JC57-14, C2, and C5. S1-specific antibodies that do notbind to RBD include G2, JC57-13, FIB_B2, and FIB_H1. G4 is an S2specific antibody.

Antibody neutralization potency was assessed by pseudotypedneutralization assay (FIG. 23) using the MERS-CoV EMC strain. Overall,the RBD-specific mAbs exhibited higher potency than S1 (non-RBD) andS2-specific mAbs. Of the tested antibodies, C2 was the most potentRBD-specific mAb, and G2 was the most potent S1-specific mAb.

Antibody neutralization breadth was assessed by pseudotypedneutralization assay. Of the RBD-specific antibodies (FIG. 24), the C2antibody neutralized 10 MERS-CoV strains with high potency including themost recent strains from Korea and China, but only partially neutralizedBisha1 (with D509G). The C5, JC57-11, and JC57-14 antibodiescross-neutralized all 8 strains with high potency, but lower than C2. Ofthe S1 (non-RBD)-specific antibodies (FIG. 25), the G2 antibodyneutralized 8 MERS-CoV strains with high potency including strains withmutations in the NTD domain of the MERS-CoV S protein. However, the A2,A10, and JC57-13 antibodies were poor neutralizers of MERS-CoV strainswith NTD mutations.

Neutralization assays using the EMC MERS-CoV strain with mutations inthe RBD domain were carried out to map the binding site of theRBD-specific antibodies. The EMC S protein was mutated as indicated inthe table below, and antibody neutralization was assayed using apseudovirus neutralization assay as described above. As shown in thetable 2, six RBD-specific mAbs can be grouped into three patterns:

1. D12, JC57-14 and C5 are interacting with similar residues, 534, 535,536 and 539

2. F11 and JC57-11 are different from other mAbs

3. C2 targets a conformation (non-linear) epitope on RBD

In the following table, “+” refers to a knock off in neutralization,“+/−” refers to a knock down in neutralization, “E” refers to enhancedneutralization, and an empty cell indicates no change in neutralization.

TABLE 2 L506F D509G T512A S532P S534A E535R E536R D539R Y540H R542GP547G N582I D12 +/− +/− + + + JC57-14 +/− + +/− +/− C5 + + + + C2 +/−+/− +/− +/− +/− +/− + +/− + F11 E + +/− +/− E +/− JC57-11 + +/− +/−

Competition binding assays were performed as described in Example 1using the identified antibodies to investigate binding patterns. Asshown in the Table 3, RBD-specific mAbs and S1 (non-RBD)-specific mAbsdo not compete binding to MERS-CoV S1 protein. RBD-specific mAbs can begrouped to 4 patterns: D12, JC57-14, C5; C2; F11; and JC57-11.S1-specific mAbs can be grouped to 2 patterns: G2, JC57-13, and FIB_H1;and A10.

TABLE 3 Competitor mAb Biotinylated mAbs (10 μg/ml) F11 D12 JC57-11JC57-14 C2 C5 G2 JC57-13 FIB_H1 A2 A10 F11 94.17 4.01 54.61 29.21 95.5354.11 10.05 10.76 10.75 17.10 21.18 D12 −3.10 97.86 52.15 88.73 97.4295.62 9.11 8.41 3.18 1.98 24.60 JC57-11 99.99 98.90 98.10 96.06 98.4996.17 26.55 21.85 22.96 7.96 27.60 JC57-14 2.51 99.20 61.29 98.15 98.4595.67 16.38 19.59 15.35 23.71 33.76 C2 77.96 62.61 39.60 48.65 98.1095.07 14.42 8.75 7.63 −13.90 3.39 C5 24.54 63.13 26.60 48.51 94.44 95.561.80 7.07 7.62 7.38 16.88 G2 15.21 7.61 18.93 28.71 2.87 29.97 86.0298.56 98.84 32.36 20.28 JC57-13 13.03 6.10 8.97 11.55 19.26 23.03 85.7295.53 97.53 52.13 52.07 FIB_H1 18.97 8.84 7.83 16.94 20.76 22.53 82.0796.49 95.41 8.34 46.42 A2 9.05 2.95 7.60 9.95 7.08 39.82 −1.37 6.89 9.371.514 3.836 A10 11.72 5.38 5.19 5.10 1614.55 28.80 3.74 7.72 4.02 85.7091.29

Mutation of C2 to Reduce Deamidation Risk.

An NG motif in the LCDR1 of C2 was mutated to reduce deamidation risk ofthe C2 antibody. The mutations tested were NG33-34NGS, NG33-34NA, andNG33-34DG. The NG33-34DG mutation decreased neutralization efficiency ofthe C2 antibody. However, the NG33-34NGS and NG33-34NA mutations did notaffect neutralization efficiency (see the Table 4).

TABLE 4 MERS CoV Strain Jordan Hasa China- Florida Korea mAb Eng1 EMC N3Buraid1 Bisha1 Batin Munich 14b GD01 USA-2 002 C2 IC50 0.0234 0.00190.0017 0.0041 0.1145 0.0037 0.0019 0.0074 0.0035 0.0117 0.0115 IC800.0569 0.0095 0.0059 0.0161 0.3361 0.0098 0.0071 0.0476 0.0112 0.03750.0190 IC90 0.1018 0.0162 0.0142 0.0278 0.6116 0.0195 0.0164 0.07700.0236 0.0599 0.0217 C2 IC50 0.0103 0.0035 0.0017 0.0049 0.1503 0.00420.0024 0.0073 0.0062 0.0049 0.0098 CDRL1- IC80 0.0371 0.0109 0.01220.0125 0.3785 0.0111 0.0098 0.0349 0.0152 0.0229 0.0259 NS IC90 0.06650.0153 0.0379 0.0249 0.6529 0.0212 0.0225 0.0867 0.0202 0.0624 0.0387 C2IC50 0.0470 0.0039 0.0015 0.0015 0.3178 0.0079 0.0018 0.0067 0.00380.0083 0.0164 CDRL1- IC80 0.0902 0.0105 0.0085 0.0148 0.9897 0.02050.0071 0.0356 0.0138 0.0484 0.0464 NA IC90 0.1085 0.0146 0.0282 0.03401.5093 0.0268 0.0164 0.0801 0.0216 0.1296 0.0670

A Human Chimeric Antibody Based on the Murine G2 Antibody

A human chimeric antibody including the V_(H) of the murine G2 antibodyand a human IgG₁ constant domain was generated by linking the G2 V_(H)to the human IgG₁ constant domain. The DNA and protein sequences of thechimeric heavy chain are provided as SEQ ID NOs: 153 and 154. Thechimeric V_(H) includes a KG-TP substitution at the beginning of theconstant domain to enhance compatibility of the human heavy chain andthe mouse light chain. The G2 mouse-human chimeric mAb (G2-hulgG KG/TP)neutralizes 11 MERS-CoV strains with comparable IC₅₀, but slightly lowerIC₈₀ and IC₉₀ to G2, as assayed using a pseudovirus neutralization assay(see the Table 5).

TABLE 5 MERS CoV Strain Jordan Hasa China Florida Korea mAb Eng1 EMC N3Buraid1 Bisha1 Batin Munich 14b GD01 USA-2 002 Mouse IC50 0.0118 0.01260.0372 0.0112 0.0119 0.0161 0.0180 0.0247 0.0202 0.0272 0.0144 G2 IC800.0432 0.0431 0.3636 0.0582 0.0317 0.0904 0.1113 0.0809 0.0592 0.06240.0367 IC90 0.0950 0.1013 2.8861 0.0903 0.0518 0.9324 0.5930 0.11000.1433 0.1059 0.0604 hIgG1- IC50 0.0140 0.0164 0.0499 0.0029 0.01290.0112 0.0457 0.1595 0.0170 0.0268 0.0153 G2 IC80 0.0807 0.0506 0.05080.1181 0.1025 KG/TP IC90 0.1258 0.1333

It will be apparent that the precise details of the methods orcompositions described may be varied or modified without departing fromthe spirit of the described embodiments. We claim all such modificationsand variations that fall within the scope and spirit of the claimsbelow.

We claim:
 1. An isolated nucleic acid molecule encoding a monoclonalantibody or an antigen binding fragment of the monoclonal antibody,wherein the monoclonal antibody or antigen binding fragment comprises aheavy chain variable region (V_(H)) and a light chain variable region(V_(H)), comprising a heavy chain complementarity determining region(HCDR)1, a HCDR2, and a HCDR3, and a light chain complementaritydetermining region (LCDR)1, a LCDR2, and a LCDR3, of the V_(H) and V_(L)set forth as any one of: (a) SEQ ID NOs: 2 and 4, respectively(JC57-13); (b) SEQ ID NOs: 6 and 8, respectively (JC57-11); (c) SEQ IDNOs: 10 and 12, respectively (JC57-14); (d) SEQ ID NOs: 36 and 38,respectively (C2); (e) SEQ ID NOs: 40 and 42, respectively (C5); (f) SEQID NOs: 44 and 46, respectively (A2); (g) SEQ ID NOs: 48 and 50,respectively (A10); (h) SEQ ID NOs: 52 and 54, respectively (FIB_B2);(i) SEQ ID NOs: 56 and 58, respectively (FIB_H1); (j) SEQ ID NOs: 36 and110, respectively (C2 LCDR1 NG-NS); (k) SEQ ID NOs: 36 and 111,respectively (C2 LCDR1 NG-NA); (l) SEQ ID NOs: 115 and 117, respectively(G2); (m) SEQ ID NOs: 119 and 121, respectively (G4); (n) SEQ ID NOs:123 and 125, respectively (D12); or (o) SEQ ID NOs: 127 and 129,respectively (F11); and wherein the monoclonal antibody or antigenbinding fragment specifically binds to MERS-CoV S protein.
 2. Thenucleic acid molecule of claim 1, wherein the HCDR1, the HCDR2, theHCDR3, the LCDR1, the LCDR2, and the LCDR3 comprise the amino acidssequences set forth as (a) SEQ ID NOs: 59, 60, 61, 62, 63, and 64,respectively (JC57-13); (b) SEQ ID NOs: 65, 66, 67, 68, 69, and 70,respectively (JC57-11); (c) SEQ ID NOs: 71, 72, 73, 74, 75, and 76,respectively (JC57-14); (d) SEQ ID NOs: 77, 78, 79, 80, 81, and 82,respectively (C2); (e) SEQ ID NOs: 83, 84, 85, 86, 87, and 88,respectively (C5); (f) SEQ ID NOs: 89, 90, 91, 92, 93, and 94,respectively (A2); (g) SEQ ID NOs: 95, 78, 96, 86, 97, and 98,respectively (A10); (h) SEQ ID NOs: 59, 99, 100, 101, 102, and 103,respectively (FIB_B2); (i) SEQ ID NOs: 104, 105, 106, 107, 108, and 109,respectively (FIB_H1); (j) SEQ ID NOs: 77, 78, 79, 112, 81, and 82,respectively (C2 LCDR1 NG-NS); (k) SEQ ID NOs: 77, 78, 79, 113, 81, and82, respectively (C2 LCDR1 NG-NA); (l) SEQ ID NOs: 130, 131, 132, 133,134, and 135, respectively (G2); (m) SEQ ID NOs: 136, 137, 138, 139,140, and 141, respectively (G4) (n) SEQ ID NOs: 142, 143, 144, 74, 145,and 146, respectively (D12); or (o) SEQ ID NOs: 147, 148, 149, 150, 151,and 152, respectively (F11).
 3. The nucleic acid molecule of claim 1,wherein the V_(H) and the V_(L) comprise the amino acid sequences setforth as: (a) SEQ ID NOs: 2 and 4, respectively (JC57-13); (b) SEQ IDNOs: 6 and 8, respectively (JC57-11); (c) SEQ ID NOs: 10 and 12,respectively (JC57-14); (d) SEQ ID NOs: 36 and 38, respectively (C2);(e) SEQ ID NOs: 40 and 42, respectively (C5); (f) SEQ ID NOs: 44 and 46,respectively (A2); (g) SEQ ID NOs: 48 and 50, respectively (A10); (h)SEQ ID NOs: 52 and 54, respectively (FIB_B2); (i) SEQ ID NOs: 56 and 58,respectively (FIB_H1); (j) SEQ ID NOs: 36 and 110, respectively (C2LCDR1 NG-NS); (k) SEQ ID NOs: 36 and 111, respectively (C2 LCDR1 NG-NA);(l) SEQ ID NOs: 115 and 117, respectively (G2); (m) SEQ ID NOs: 119 and121, respectively (G4); (n) SEQ ID NOs: 123 and 125, respectively (D12);or (o) SEQ ID NOs: 127 and 129, respectively (F11).
 4. The nucleic acidmolecule of claim 1, comprising a human framework region.
 5. The nucleicacid molecule of claim 1, encoding the monoclonal antibody.
 6. Thenucleic acid molecule of claim 5, wherein the antibody is a humanizedantibody comprising a human constant domain.
 7. The nucleic acidmolecule of claim 6, wherein the heavy chain of the antibody comprisesthe amino acid sequence set forth as SEQ ID NO:
 154. 8. The nucleic acidmolecule of claim 5, wherein the antibody is an IgG.
 9. The nucleic acidmolecule of claim 5, comprising a recombinant constant domain comprisinga modification that increases the half-life of the antibody.
 10. Thenucleic acid molecule of claim 9, wherein the modification increasesbinding to the neonatal Fc receptor.
 11. The nucleic acid molecule ofclaim 9, wherein the recombinant constant domain is an IgG₁ constantdomain comprising M428L and N434S mutations.
 12. The nucleic acidmolecule of claim 1, encoding the antigen binding fragment of themonoclonal antibody.
 13. The nucleic acid molecule of claim 12, whereinthe antigen binding fragment is a Fv, Fab, F(ab′)₂, scFV or a scFV₂fragment.
 14. The nucleic acid molecule of claim 1, comprising a cDNAmolecule encoding the antibody or antigen binding fragment.
 15. Thenucleic acid molecule of claim 1, operably linked to a promoter.
 16. Anexpression vector comprising the nucleic acid molecule of claim
 1. 17. Ahost cell, comprising the nucleic acid molecule of claim
 1. 18. Apharmaceutical composition, comprising the nucleic acid molecule ofclaim 1, or a vector comprising the nucleic acid molecule; and apharmaceutically acceptable carrier.
 19. The pharmaceutical compositionof claim 17, wherein the composition is sterile.
 20. The pharmaceuticalcomposition of claim 18, wherein the composition is in unit dosage formor a multiple thereof.
 21. The pharmaceutical composition of claim 20,comprising: a first nucleic acid molecule encoding an antibodycomprising a heavy chain variable region and a light chain variableregion, comprising a HCDR1, a HCDR2, and a HCDR3, and a LCDR1, a LCDR2,and a LCDR3, of the V_(H) and V_(L) set forth as SEQ ID NOs: 36 and 38,respectively (C2), wherein the monoclonal antibody specifically binds toMERS-CoV S protein; and a second nucleic acid molecule encoding anantibody comprising a heavy chain variable region and a light chainvariable region, comprising a HCDR1, a HCDR2, and a HCDR3, and a LCDR1,a LCDR2, and a LCDR3, of the V_(H) and V_(L) set forth as SEQ ID NOs:115 and 117, respectively (G2), wherein the monoclonal antibodyspecifically binds to MERS-CoV S protein.
 22. The pharmaceuticalcomposition of claim 21, wherein: the HCDR1, the HCDR2, the HCDR3, theLCDR1, the LCDR2, and the LCDR3 of the first isolated monoclonalantibody comprise the amino acids sequences set forth as SEQ ID NOs: 77,78, 79, 80, 81, and 82, respectively; and the HCDR1, the HCDR2, theHCDR3, the LCDR1, the LCDR2, and the LCDR3 of the second isolatedmonoclonal antibody comprise the amino acids sequences set forth as SEQID NOs: 130, 131, 132, 133, 134, and 135, respectively.
 23. Thepharmaceutical composition of claim 22, wherein: the V_(H) and the V_(L)of the first isolated monoclonal antibody comprise the amino acidsequences set forth as SEQ ID NOs: 36 and 38, respectively (C2); and theV_(H) and the V_(L) of the second isolated monoclonal antibody comprisethe amino acid sequences set forth as SEQ ID NOs: 115 and 117,respectively (G2).
 24. The pharmaceutical composition of claim 23,wherein: the heavy chain of the second isolated monoclonal antibodycomprises an amino acid sequence set forth as SEQ ID NO:
 154. 25. A kitcomprising a container comprising the nucleic acid molecule of claim 1,and instructions for using the kit.
 26. A method of producing anantibody or antigen binding fragment that specifically binds to MERS-CoVS protein, comprising: expressing the nucleic acid molecule of claim 1in a host cell, thereby producing the antibody or antigen bindingfragment.
 27. A method of inhibiting a MERS-CoV infection in a subject,comprising administering to a subject with or at risk of a MERS-CoVinfection an effective amount of the pharmaceutical composition of claim18.